and saturate the filtrate with sulphuretted hydrogen till all excess lead is removed.
Filter off" the lead sulphide, neutralize the filtrate to phenolphthalein with ammonia,
and evaporate to half bulk or less on a water-bath, when the inulin will probably
v] HYDROLYZING ENZYMES 59
begin to deposit, Then pour into an equal volume of alcohol, and allow to stand for
one or two days.
The crude precipitate of inulin is filtered off, dissolved in a small
amount of water, and reprecipitated with alcohol. It can be washed with alcohol
and ether and dried over sulphuric acid.
The Artichoke (Helianthus tuberosus) may also be used, about 12 tubers being
Expt. 60. Tests for inulin. Make a solution of some of the inulin prepared in
Expt. 59 in hot water. It will readily dissolve giving a clear solution. With the
solution make the following tests :
(a) Make a very dilute solution of iodine and add to it a drop or two of iuulin
solution : the brown colour will be unaffected.
(6) Boil some inulin solution with a little Fehling : no reduction takes place.
If the inulin solution which is being used should reduce Fehling it indicates that
sugar is present as impurity. If this is the case, then a little of the solid inulin
should be washed free from sugar by means of alcohol before proceeding with the
(c) To a little inulin solution add some 1 % alcoholic solution of a-naphthol and
a few drops of concentrated sulphuric acid and warm. A deep violet colour is
produced. This is due to the formation of furfural from the laevulose produced in
hydrolysis (see laevulose, p. 51).
(d) To a little inulin solution add about an equal quantity of strong hydrochloric
acid and a few crystals of resorcin. A red coloration is formed. This reaction
(Seliwanoff 's test) is also due to the presence of laevulose (see laevulose, p. 51).
Expt. 61. Hydrolysis of inulin. Some inulin is dissolved in very dilute hydrochloric
acid (about 0'5 %) and heated on a water-bath for half an hour in a round-bottomed
flask provided with an air condenser (see p. 45). The solution is then neutralized
with sodium carbonate and concentrated on a water-bath. With the concentrated
solution make the following tests :
() Boil with a little Fehling : the solution is rapidly reduced.
(6) Make the osazone test (see p. 49). Glucosazone crystals will be found to be
formed on microscopic examination. (Laevulose forms the same osazone as glucose.)
(c) Make the tests (c) and (d] of the last experiment. A positive result will be
given in each case.
The mannans which have already been mentioned (see p. 50) are
condensation products of the hexose, mannose. They occur most fre-
quently, either mixed, or in combination, with the condensation products
of other hexoses and pentoses (glucose, galactose, fructose and arabinose)
as galacto mannans, glucomannans, fructomannans, mannocelluloses, etc.
Such mixtures or compounds of which mannans form a constituent are
widely distributed in the seeds of many plants, i.e. Palms (including the
Date-palm), Asparagus (Ruscus), Clover (Trifoliuin), Coffee Bean (Coffea
arabica), Onion (Allium Cepa) and of members of the Legurninosae,
60 CARBOHYDRATES AND THEIR [CH.
Rubiaceae, Coniferae and Umbelliferae. In seeds the mannans may
constitute, together with cellulose, the thickened cell-walls of the endo-
sperm and are included in the term " reserve- or hemi-cellulose " though
they are not strictly celluloses. " Vegetable ivory," which is the endo-
sperm of the Palm, Phytelephas macrocarpa, contains considerable
quantities of a mannan and is used as a source of mannose. Mannans,
in addition, form constituents of certain mucilages, as for instance those
in Lily bulbs (Lilium candidum, L. bulbiferum, L. Martagon and others)
(Parkin, 23) and tubers of various genera of the Orchidaceae : they are
also found in the roots of the Dandelion (Taraxacum), Helianthus and
Chicory, Asparagus and Clover, and in the wood and leaves of various
Many of the mannans, unlike true celluloses, are readily hydrolyzed
by dilute hydrochloric and sulphuric acids. The mannan in the Coffee
Bean, however, is hydrolyzed with difficulty.
These substances bear the same relationship to the hexose, galactose,
as the mannans to mannose, that is, they are condensation products of
galactose (see p. 49). Similarly they frequently occur, together with
the condensation products of other sugars, as galactoaraban, galactoxylan,
galactomannan, etc. As such they form constituents of many gums and
mucilages and of the cell-walls of the reserve tissue of seeds, i.e. the
Coffee Bean (Goffea arabicd), the Bean (Faba), the Lupin (Lupinus), the
Paeony (Paeonia), the Kidney Bean (Phaseolus), the Date (Phoenix),
the Pea (Pisum), the Nasturtium (Tropaeolum) and many others (Schulze,
Steiger and Maxwell, 29).
These substances occur widely distributed among plants, especially
trees. Some gums are wholly soluble in water giving sticky colloidal
solutions : others are only partially soluble. They are all insoluble in
alcohol. In the solid state they are translucent and amorphous.
Chemically the gums are varied in nature ; they may in general be
regarded as consisting of complex acids in combination 1 with condensa-
tion products of various sugars, such as araban, xylan, galactan, etc.
On hydrolysis they give mixtures of the corresponding sugars, arabinose,
xylose, galactose, etc., in varying proportions, though in some cases one
v] HYDROLYZING ENZYMES 61
Some of the best-known gums are the following :
Gum Arabic (arabin). This substance is obtained from an Acacia
(Acacia Senegal), a native of the Soudan. The gum exudes from the
branches. Other species of Acacia yield inferior gums. Gum arabic is
a mixture of the calcium, magnesium and potassium salts of arabic acid,
a weak acid of which the constitution is unknown, in combination with
araban and galactan.
Gum Tragacanth. This is a product from several Tragacanth shrubs
which are species of Astragalus (Leguminosae), chiefly A. gummifer. It
is obtained by wounding the stem and allowing the gum to exude and
harden. On hydrolysis it gives a mixture of complex acids and various
sugars such as arabinose, galactose and xylose.
Cherry Gum (cerasin) occurs in the wood of the stems and branches
of the Cherry (Prunus Cerasus), the Bird Cherry (P. Padus), the Plum
(P. domestica), the Almond (P. Amygdalus) and other trees of the
Rosaceae. It exudes from fissures of the bark. On hydrolysis it yields
almost entirely arabinose.
Expt. 62. Reactions of Gum Arabic. Put a little gum arabic into an evaporating
dish and add a little water. Heat gently and stir. The gum will slowly dissolve,
giving a thick sticky solution which does not solidify or gel on cooling. Make the
following tests, using a little of the gum solution in a test-tube each time.
(a) Add a little alcohol. The gum is precipitated.
(6) Add a little Fehling's solution and boil. No reduction takes place.
The three following experiments show the presence of pentosan complexes in the
gum (see also Expt. 38, p. 44):
(c) Add a little phloroglucin to the gum and then strong hydrochloric acid. No
colour is produced. Now heat, and a cherry-red colour appears.
(cT) Heat the gum solution with a little concentrated hydrochloric acid and then
add a trace of orcinol. Warm again and then add one or two drops of strong ferric
chloride solution. A green coloration will be produced.
(e) Heat the gum solution strongly with hydrochloric acid, and, after heating for
a few minutes, place a piece of niter-paper soaked in a solution of aniline acetate in
the mouth of the test-tube. A cherry-red coloration indicative of furfural will be
Expt. 63. Hydrolysis of Gum Arabic. Weigh out 10 gms. of gum arabic. Put it
into a round-bottomed flask and add 100 c.c. of water and 4 c.c. of strong sulphuric
acid. Warm gently until the gum goes into solution. Then fit the flask with an
air condenser (see p. 45) and heat on a water-bath for about 4 hrs. Cool the
solution, and neutralize with barium carbonate. Filter off" the barium sulphate and
concentrate the solution on a water-bath. Boil a drop or two of the syrup with
Fehling's solution and show that reduction takes place. (The original gum either
does not reduce Fehling at all, or, if so, only slightly.) Then add a little nitric acid
62 CARBOHYDRATES AND THEIR [CH.
(sp. gr. 1'15, see Expt. 43) to the syrup and heat on a water-bath almost to dryness.
Pour the residue into about 100 c.c. of water and allow to stand. A microcrystalline
precipitate of mucic acid is formed showing the presence of galactose (see p. 50) as
a product of hydrolysis.
The characteristic of these substances is that they swell up in water
and produce colloidal solutions which are slimy.
Mucilages are widely distributed and may occur in any organ of the
plant. Sometimes they are confined to certain cells, mucilage sacs or
canals. They are distinguished from the pectic substances by the fact
that they do not gelatinize. Some of the best known examples of
mucilage-containing tissues are those in the root and flower of the
Hollyhock (Althaea rosed) : in succulent plants (Aloe, Euphorbia), in
bulbs (Scilla, Allium) and tubers (Orchis Morio): in seeds of Flax or
Linseed (Linum) and in fruits of Mistletoe (Viscum album}.
The mucilages vary in composition. They appear to be largely, if
not wholly, condensation products of various sugars (galactose, mannose,
glucose, xylose, arabinose), similar constituents to those of many gums
and hemicelluloses. On hydrolysis various mixtures of sugars are pro-
duced. Of the mucilages, that from linseed has been thoroughly
investigated. It has been found on hydrolysis to give sugars only, e.g.
arabinose, xylose, glucose and galactose. In this respect mucilages differ
from gums, since the latter have always some other accompanying sub-
stance in addition to sugars.
Expt. 64. Preparation and properties of mucilage from Linseed (Linum) (Neville,
21). Take about 60 gms. of linseed and let it soak for 24 hrs. in 300 c.c. of water.
Then separate the slime from the seeds by squeezing through muslin, and add to the
liquid about twice its volume of 96-98 % alcohol. The mucilage is precipitated as
a thick slimy precipitate. Filter off the precipitate and wash with alcohol. By
washing with absolute alcohol and ether and finally drying in a desiccator, the
mucilage may be obtained as a powder.
Add water to some of the mucilage. It swells up and finally gives an opalescent
solution. Make with it the following tests :
(a) Add iodine. No colour is given.
(b) Add a little Fehling's solution and boil. No reduction takes place.
Expt. 65. Hydrolysis of Linseed mucilage. Put the remainder of the mucilage in
a round-bottomed flask and add 50 c.c. of 4 % sulphuric acid. Fit the flask with
an air condenser (see p. 45) and heat for at least four hours on a water-bath.
Cool and neutralize with barium carbonate. Filter off the barium sulphate, and
v] HYDROLYZING ENZYMES 63
concentrate the filtrate on a water- bath. With the concentrated solution make the
following tests :
(a) Add a few drops to a little boiling Fehling solution. Reduction immediately
(6) Make the phloroglucin, orcinol and furfural tests for pentoses, using a small
quantity only of the hydrolysis mixture for the tests. A positive result will be given
in each case. The pentoses, arabinose and xylose, are responsible for these reactions.
(c) Add to some of the solution phenylhydrazine hydrochloride, sodium acetate
and a little acetic acid, and leave in boiling water for half an hour for the osazone test
[see Expt. 41 (d)\ A mixture of osazones will separate out, among which glucosazone
can be identified.
(d) Concentrate the remainder of the solution and then add some nitric acid of
sp. gr. T15 (see Expt. 43). Evaporate down on a water-bath to one-third of the bulk
of the liquid and then pour into about 100 c.c. of water. A white microcrystalline
precipitate of mucic acid will separate out, either at once or in the course of a day or
two. This demonstrates the presence of galactose.
PEC TIC SUBSTANCES.
These substances are considered at this point since they are said to
constitute, in more or less intimate connexion with cellulose, the middle
lamella of cell-walls in many tissues. The pectic substances are fre-
quently found in the juices of succulent fruits in which the tissues have
disintegrated, such as red currants and gooseberries. They have been
isolated chiefly from fleshy roots, stems or fruits, as, for instance, from
turnips, beetroot, rhubarb stems, apples, cherries and strawberries.
Recent work (Schryver and Haynes, 28) points to the fact that in
turnips, strawberries, rhubarb stems and apples, there is the same pectic
material, and it is possible that all such substances may be identical.
The compound isolated in the above case is of an acidic nature and has
been termed pectinogen. When pectinogen is treated with dilute
solutions of caustic alkali at ordinary temperatures, it is rapidly changed
into a second substance termed pectin, which is readily converted into
a gel under certain conditions.
In the case of juicy fruits, such as currants and gooseberries, the
pectinogen can be precipitated as a gelatinous precipitate by adding
alcohol to the expressed juice. In the case of fleshy fruits, stems and
roots, the procedure is as follows. The tissues are thoroughly dis-
integrated in a mincing machine and pressed free from all juice in a
powerful press. The residue is then dried, finely ground, washed with
water and finally extracted with dilute ammonium oxalate solution in
which pectinogen is soluble. The extract is concentrated and the
64 CARBOHYDRATES AND THEIR [CH.
pectinogen precipitated by alcohol. It may be purified by reprecipita-
Pectinogen is precipitated from aqueous solution by alcohol as a very
bulky gelatinous mass, but when dried it forms an almost colourless granu-
lar powder. Put into water it absorbs large quantities of liquid and dis-
solves slowly, giving an opalescent solution with a distinctly acid reaction.
As mentioned above pectinogen in alkaline solution is rapidly con-
verted into pectin. A solution of pectinogen is not precipitated either
by acid or dilute solutions of calcium salts but, after treatment with
alkali and conversion into pectin, both the aforesaid reagents produce
gelatinous precipitates. A similar precipitate is also formed when lime
water is added in excess to a solution of pectinogen and it is allowed to
stand. There is little doubt that the pectinogen is converted by the
alkali into pectin. Pectin is also an acid substance and it is insoluble
in water, giving an insoluble salt with calcium. After treatment of
pectinogen with alkali the pectin can, as already stated, be precipitated
by adding acid.
Analyses of pectin from various sources have led to the suggestion
of C^H^Oje as its formula. There is also evidence that it contains one
pentose group. This can be detected and estimated by the furfural
phloroglucide method (see Expt. 53).
Expt. 66. Extraction and reactions of pectinogen. Take about half a pound of red
currants and squeeze out the juice through fine muslin into a large beaker. Then add
to the juice about 2-3 times its bulk of 96-98% alcohol. A bulky gelatinous precipi-
tate of pectinogen will separate out. Allow the precipitate to stand for a time in the
alcohol, and then filter off. Wash with alcohol and finally press free from liquid.
Dissolve the precipitate in as little water as will enable it to go into solution. To two
small portions of the solution add respectively (a) a few drops of strong hydrochloric
acid, (6) an excess of calcium chloride solution. Note that no precipitate is formed in
Expt. 67. Conversion of pectinogen into pectin, and reactions of pectin. Take about
one-third of the pectinogen solution prepared in Expt. 66, make it alkaline with
caustic soda, and let it stand for about 10-15 minutes. Then divide the solution
into two parts and add respectively (a) sufficient hydrochloric acid to acidify,
(6) excess of calcium chloride solution. In the first case a gel of pectin is formed :
in the second case a gelatinous precipitate of the calcium salt of pectin.
To a further quantity of the pectinogen add excess of lime water and let it stand.
The gelatinous calcium precipitate will separate out in a short time.
Expt. 68. Detection of the pentose group in pectinogen. Filter off the pectin gel
obtained in the last experiment and allow it to dry. Then test for the pentose group
by the orcinol, phloroglucinol and furfural tests (see Expt. 38). All results will be
found to be positive.
v] HYDROLYZING ENZYMES 65
The extraction of pectinogen, etc. in the above experiments can equally well be
carried out with other material, e.g. ripe gooseberries, raspberries and strawberries,
using exactly the same methods.
Expt. 69. Preparation of pectinogen from Turnips. Take two full-sized turnips
and mince them finely in a mincing machine. Then wrap the mass in a piece of
strong unbleached calico and press out the juice as completely as possible in a press.
The juice contains little pectinogen and can be thrown away. The pressed mass is
then thrown into 0'5 / ammonium oxalate solution heated to 80-90 C. on a water-
bath and stirred to make a paste. The liquid is again rapidly pressed out in the press.
To the viscid extract an equal volume of 96 % alcohol is added, and the pectinogen
separates out as a voluminous gelatinous precipitate. This is filtered off and, when
pressed free from alcohol and dried, can be used for tests as in the previous experi-
The gelatinization of pectinogen can also be brought about by certain
enzymes termed pectases which are found in the juices of various plants,
i.e. root of Carrot (Daucus Carota) and leaves of Lucerne (Medicago
sativa), Lilac (Syringa vulgaris) and Clover (Trifolium pratense).
Expt. 70. Action of pectase on pectinogen. Make an extract of either Lucerne or
Clover leaves by pounding them in a mortar with a little water, and then filter. Add
the filtrate to some of the pectinogen solution prepared in Expt. 66 or 69. On
standing a gelatinous precipitate will be produced. Should the reaction be slow, it
may be accelerated by placing the mixture in an incubator.
Celluloses are very important polysaccharides. They form constituents
of the structural part of all the higher plants. The cell-wall of the
young cell consists entirely of cellulose, but in older cells the walls may
be lignified, cuticularized, etc., i.e. the cellulose may be accompanied by
other substances such as lignin, cutin, mucilage, etc. In the light of
these facts the term cellulose is made to include :
1. Normal celluloses.
2. Compound celluloses.
(c) Adipo- or cuto-celluloses.
3. Pseudo- or Reserve celluloses.
True or normal cellulose. Of this substance, as we have said,
many cell- walls are composed. The most familiar form of cellulose is
cotton, which consists of hairs, each being a very long empty cell, from
the testa or coat of the seed of the Cotton plant (Gossypium herbaceum).
66 CARBOHYDRATES AND THEIR [CH.
Crude cotton (i.e. the hair cell-walls) is not quite pure cellulose, but
contains a small amount of impurity from which it is freed by treatment
first with alkali and subsequently with bromine or chlorine. All kinds
of cotton material, cotton-wool, and the better forms of paper (including
filter-paper) may be regarded as almost pure cellulose.
Pure cellulose is a white, somewhat hygroscopic, substance. It is
insoluble in water and all the usual solvents for organic substances. It
is, however, soluble in a solution of zinc chloride in hydrochloric acid in
the cold, and in a solution of zinc chloride alone on warming. It is also
soluble in ammoniacal cupric oxide (Schweizer's reagent).
In addition cellulose is soluble in concentrated sulphuric acid, which
on standing, converts it first into a hydrate and then finally into
glucose. If, however, water is added to the sulphuric acid solution as
soon as it is made, the gelatinous hydrate of cellulose is precipitated.
This substance is termed " amyloid " since it gives a blue colour with
iodine. Concentrated nitric acid converts cellulose into nitrates, of
which one is the substance, gun-cotton. In 10 / alkalis cotton fibres
thicken and become more cylindrical. This procedure has been em-
ployed by Mercer to give a silky gloss to cotton, and the resultant
product is called mercerized cotton.
Expt. 71. The colour tests and solubilities of cellulose.
(a) Dip a little cotton-wool into absolution of iodine in potassium iodide. Then
put the stained wool into an evaporating dish and add a drop or two of concentrated
sulphuric acid. A blue coloration is given. This is due to the formation of the
hydrate " amyloid " mentioned above.
(6) Dip some cotton-wool into a calcium chloride iodine solution. (To 10 c.c. of
a saturated solution of calcium chloride add 0'5 gm. of potassium iodide and O'l gm.
of iodine. Warm gently and filter through glass-wool.) A rose-red coloration is
produced which eventually turns violet.
(c) Heat a strong solution of zinc chloride (6 pts. of zinc chloride to 10 pts. of
water) in an evaporating dish and add 1 part of cotton-wool. The cellulose will in
time become gelatinized, and if a little water is added from time to time, a solution
will eventually be obtained on continuous heating.
(d) Make a solution of zinc chloride in twice its weight of concentrated hydro-
chloric acid and add some cotton- wool. The wool will rapidly go into solutiQii in the
(e) Add some cotton-wool to an ammoniacal copper oxide solution and note that
it dissolves. (To a strong solution of copper sulphate add some ammonium chloride
and then excess of caustic soda. Filter off the blue precipitate of cupric hydroxide,
wash well, dry thoroughly, and dissolve in strong ammonia.) Add strong hydrochloric
acid and the cellulose is precipitated out again. Then add water and wash the
precipitate until it is colourless. Test the roughly dried precipitate with a little
iodine and strong sulphuric acid. A blue coloration is given.
v] HYDROLYZING ENZYMES 67
All the above tests may be repeated with threads from white cotton material,
with filter-paper and good white writing paper.
Try tests (a) and (6) with newspaper, and note that they are not so distinct as
with writing paper owing to the presence of ligno-cellulose (see Expt. 73).
Expt. 72. Hydrolysis of cellulose by acid. Dissolve as much filter-paper as possible
in 5 c.c. of concentrated sulphuric acid and when all is in solution pour into 100 c.c.
of distilled water. Boil the solution in a round-bottomed flask fitted with an air
condenser (see p. 45) and use a sand-bath for heating. After boiling for an hour,
cool and neutralize the solution with solid calcium carbonate. Add a little water if
necessary and filter. Test the filtrate with the following tests :
(a) Make the osazone [see Expt. 41 (c?)]. Note that crystals of glucosazone are
(6) Add a little Fehling's solution and boil. . Note that reduction takes place.
Instead of using filter-paper, the above experiment may also be carried out with
cotton-wool or threads from white cotton material.
Ligno-cellulose. As the cells in plants grow older the walls usually
become lignined, that is part of the cellulose becomes converted into
ligno-cellulose. The extreme amount of change is found in wood. The
least amount in such fibres as those from the stem of the Flax (Linum
mitatissimum) which, when freed from such impurities, consist of cellu-
lose only and constitute linen. Other fibres, containing more ligno-
cellulose, are those of the stem of the Hemp plant (Cannalris sativa)
and the Jute plant (Corchorus) from which string, rope, canvas, sacking
and certain carpets are made. The percentages of pure cellulose in
these various lignified tissues are as follows :
Cotton fibre 88'3%
Flax and Hemp fibre ... 72-73 %
Beech and Oak wood ... 35-38 %
The ligno-celluloses are generally regarded as consisting of cellulose
and two other constituents, of which one contains an aromatic nucleus
and the other is of the nature of a pentosan (see xylan, p. 53). Both
are sometimes classed together and termed lignin or lignon. The lignin
reactions (see below) depend on the presence of an aromatic complex.