=392. Preliminary Treatment.=—The chief disturbing components of
vegetable tissues, in respect of their influence on the separation
and estimation of the proteid constituents, are fats and oils and
coloring matters. In many cases these bodies are present in such
small quantities as to be negligible, as, for instance, in rice. In
other cases they exist in such large proportions as to present almost
insuperable difficulties to analytical operations, as is the case with
oily seeds. In all instances, however, it is best to remove these
bodies, even when present in small proportions, provided it can be done
without altering the character of the proteid bodies. This is secured
by extracting the fine-ground vegetable material first with petroleum
ether, and afterwards with strong alcohol and ether. Practically, all
of the fatty bodies and the greater part of the most objectionable
coloring matters are removed by this treatment. The extraction should
in all cases be made at low temperatures, not exceeding 35°, to avoid
the coagulating effect of higher temperatures upon the albuminous
bodies which may be present.
In this laboratory, fatty seeds, as for instance peanuts, are first
ground into coarse meal, then extracted with petroleum ether, ground
to a fine meal and the fat extraction completed with petroleum ether,
ninety-five per cent alcohol and pure sulfuric ether. The residue of
the last solvent may be removed by aspirating air through the extracted
meal. In some cases, it is advisable to extract with ethyl ether before
as well as after the alcoholic extraction. This treatment removes at
least a part of the water and prevents the dilution of the first part
of alcohol added to such an extent as to make it dissolve some of the
proteid matters. In each case, a portion of the alcoholic extract
should be tested qualitively for proteid matter. If any be found,
stronger alcohol should be used for, at least, the first extraction. A
portion of the meal, prepared as above directed, is extracted with a
ten per cent solution of sodium chlorid, as described further on, and a
measured portion of the filtered extract diluted with water until the
proteid matter in solution begins to be precipitated. By this treatment
the proper strength of the salt solution, to be used for the subsequent
extraction, is determined. To save time in dialyzing, the solution of
salt employed as a solvent should be as dilute as possible.
The mixture of meal and solvent sometimes filters with difficulty.
In these cases, it is advisable to first pour it into a linen bag
from which the liquid portion can be removed by gentle pressure and
subsequently filtered through paper. As a last resort, the liquid
secured from the linen filter can be saturated with ammonium, zinc or
magnesium sulfate, whereby all the proteid matters are thrown out.
After filtering, the residue is again dissolved in salt solution and
can then be readily filtered through paper.
The clear filtrate should be tested by fractional precipitation by heat
and the final filtrate by acetic acid, as will be described further on.
The proteid matter may be further freed from amid compounds by
treatment with copper sulfate.[365] This treatment is not advisable,
however, except for the purpose of determining the total proteid
nitrogen in the sample. The action of the water, heat and cupric
sulfate combined is capable of inducing grave changes in the character
of the residual matter which would seriously interfere with the results
of subsequent studies of the nature of the proteid bodies.
In many instances, as with cereal grains, the separation of the proteid
bodies is accomplished by no further preliminary treatment than is
necessary to reduce them to the proper degree of fineness.
=393. Diversity of Character.=—The proteids which occur in vegetable
products are found in all parts of the tissues of the plants, but in
cereals especially in the seeds. In grass crops and in some of the
legumes, such as clover, the nitrogenous matters are chiefly found in
the straw and leaves. The general classification of these bodies has
already been given, but each kind of plant presents marked variations,
not only in the relative proportions of the different classes, but also
in variations in the nature of each class. For this reason the study
of vegetable proteids is, in some respects, a new research for each
kind of plant examined. There are, however, some general principles
which the analyst must follow in his work, and an attempt will be made
here to establish these and to construct thereon a rational method
of conducting the investigation. In the separation and estimation
of complex bodies so nearly related to each other, it is difficult
not only to secure satisfactory results, but also to prevent the
transformation of some forms of proteid matter into others nearly
related thereto by the action of the solvents used for separation and
precipitation.
=394. Separation of Gluten from Wheat Flour.=—The most important
proteid in wheat is the body known as gluten, a commercial name given
to the nitrogenous matters insoluble in cold water. The gluten thus
obtained does not represent a single chemical compound, but is a
complex consisting of at least two proteid bodies, which together form
an elastic, pasty mass, insoluble in cold water containing a trace
of mineral salts. This mass has the property of holding mechanically
entangled among its particles bubbles of gas, which, expanding under
the action of heat during cooking, give to bread made of glutenous
flours its porous property.
In respect of proteids, the American wheats, as a rule, are quite
equal to those of foreign origin. This is an important characteristic
when it is remembered that both the milling and food values of a wheat
depend largely on the nitrogenous matter which is present. It must
not be forgotten, however, that merely a high percentage of proteids
is not always a sure indication of the milling value of a wheat. The
percentage of gluten to the other proteid constituents of a wheat is
not always constant, and it is the gluten content of a flour on which
its bread making qualities chiefly depend. The percentage of moist
gluten gives, in a rough way, the property of the glutenous matter of
absorbing and holding water under conditions as nearly constant as
can be obtained. In general, it may be said that the ratio between
the moist gluten and the dry gluten in a given sample is an index for
comparison with other substances in the same sample. Upon the whole,
however, the percentage of dry gluten must be regarded as the safer
index of quality. In respect of the content of glutenous matter, our
domestic wheats are distinctly superior to those of foreign origin.
They are even better than the Canadian wheats in this respect. It may
be fairly inferred, therefore, that while our domestic wheats give a
flour slightly inferior in nutritive properties to that derived from
foreign samples, it is nevertheless better adapted for baking purposes,
and this quality more than compensates for its slight deficiency in
respect of nutrition, a deficiency, which, however, is so minute as to
be hardly worth considering.[366]
The gluten is separated in this laboratory from the other constituents
of a flour by the following process:
Ten grams of the fine-ground flour are placed in a porcelain dish,
well wet with nearly an equal weight of water at a temperature of not
to exceed 15°, and the mass worked into a ball with a spatula, taking
care that none of it adheres to the walls of the dish. The ball of
dough is allowed to stand for an hour, at the end of which time it
is held in the hand and kneaded in a stream of cold water until the
starch and soluble matter are removed. The ball of gluten thus obtained
is placed in cold water and allowed to remain for an hour when it is
removed, pressed as dry as possible between the hands, rolled into a
ball, placed in a flat bottom dish and weighed. The weight obtained
is entered as moist gluten. The dish containing the ball of gluten is
dried for twenty hours in a steam-bath, again weighed, and the weight
of material obtained entered as dry gluten. The determination of dry
and moist gluten cannot in any sense be regarded as an isolation and
estimation of a definite chemical compound. For millers’ and bakers’
purposes, however, the numbers thus obtained have a high practical
value. A typical wheat grown in this country will contain about 26.50
per cent of moist and 10.25 per cent of dry gluten.
The gluten of wheat is composed of two proteid bodies, gliadin and
glutenin.[367] Gliadin contains 17.66 per cent, and glutenin 17.49 per
cent of nitrogen. Gliadin forms a sticky mass when mixed with water and
is prevented from passing into solution by the small content of mineral
salts present in the flour. It serves to bind together the other
ingredients of the flour, thus rendering the dough tough and coherent.
Glutenin serves to fix the gliadin and thus to make it firm and solid.
Glutenin alone cannot yield gluten in the absence of gliadin, nor
gliadin without the help of glutenin. Soluble metallic salts are also
necessary to the formation of gluten, and act as suggested above, by
preventing the solution of the gliadin in water, during the process of
washing out the starch. No fermentation takes place in the formation of
gluten from the ingredients named.
The gluten, which is obtained in an impure state by the process above
described, is, therefore, not to be regarded as existing as such in the
wheat kernel or flour made therefrom, but to arise by a union of its
elements by the action of water.
=395. Extraction with Water.=—It is quite impossible to get an extract
from fine-ground vegetable matter in pure water because the soluble
salts of the sample pass at once into solution and then a pure water
solvent becomes an extremely dilute saline solution. The aqueous
extract may, however, be subjected to dialysis, whereby the saline
matter is removed and the proteid matter, not precipitated during the
dialytic process, may be regarded as that part of it in the original
sample soluble in pure water. Nevertheless, in many instances, it
is important to obtain an extract with cold water. In oatmeal the
aqueous extract is obtained by Osborne as follows:[368] Five pounds of
fine-ground meal are shaken occasionally with six liters of cold water
for twenty-four hours, the liquid removed by filtration and pressure
and the extraction continued with another equal portion of water in
the manner noted. The two liquid extracts are united and saturated
with commercial ammonium sulfate which precipitates all the dissolved
proteid matter. The filtrate obtained is collected on a filter, washed
with a saturated solution of ammonium sulfate and removed as completely
as possible from the filter paper by means of a spatula. Any residual
precipitate remaining on the paper is washed into the vessel containing
the removed precipitate and the undissolved precipitate well beaten up
in the liquid, which is placed in a dialyzer with a little thymol, to
prevent fermentation, and subjected to dialysis for about two weeks. At
the end of that time, the contents of the dialyzer are practically free
of sulfates. The contents of the dialyzers are then thrown on a filter
and in the filtrate are found those proteids first extracted with
water, precipitated with ammonium sulfate and redissolved from this
precipitated state by pure water. In the case of oatmeal, this proteid
matter is not coagulated by heat, and may be obtained in the dry state
by the evaporation of the filtrate last mentioned at a low temperature
in vacuo. It is evident that the character of the proteid matter thus
obtained will vary with the nature of the substance examined. In the
case of oats, it appears to be a proteose and not an albumin.
=396. Action of Water on Composition of Proteids.=—When a body, such
as oatmeal, containing many proteids of nearly related character, is
exposed to the action of a large excess of water, the proteid bodies
may undergo important changes whereby their relations to solvents are
changed. After oatmeal has been extracted with water, as described
above, the proteid matter originally soluble in dilute alcohol
undergoes an alteration and assumes different properties. The same
remark is applicable to the proteid body soluble in dilute potash.
Nearly all the proteid matter of oatmeal is soluble in dilute potash,
if this solvent be applied directly, but if the sample be previously
treated with water or a ten per cent salt solution the subsequent
proportion of proteid matter soluble in dilute potash is greatly
diminished.[369] Water applied directly to the oatmeal apparently
dissolves an acid albumin, a globulin or globulins, and a proteose. The
bodies, however, soluble in water, exist only in small quantities in
oatmeal. Experience has shown that in most instances, it is safer to
begin the extraction of a cereal for proteid matter with a dilute salt
solution rather than with water, and to determine the matters soluble
in water alone by subsequent dialysis.
=397. Extraction with Dilute Salt Solution.=—In general, it is
advisable to begin the work of separating vegetable proteids by
extracting the sample with a dilute brine usually of ten per cent
strength. As conducted by Osborne and Voorhees, on wheat flour, the
manipulation is carried on as follows:[370]
The fine-ground whole wheat flour, about four kilograms, is shaken with
twice that weight of a ten per cent sodium chlorid solution, strained
through a sieve, to break up lumps, and allowed to settle for sixteen
hours. At the end of this time, about half of the supernatant liquid
is removed by a siphon or by decantation and filtered. Two liters
more of the salt solution are added, the mixture well stirred and the
whole brought onto the filter used above. The filtrate is collected
in successive convenient portions and each portion, as soon as it is
obtained, is saturated with ammonium sulfate. All the proteid matter is
precipitated by this reagent. The precipitate is collected on a filter,
redissolved in a convenient quantity of the salt solution and dialyzed
for fourteen days or until all sulfates and chlorids are removed. The
proteid matter, which is separated on dialysis, in this instance, is a
globulin.
The proteid matter not precipitated on dialysis is assumed to be that
part of the original substance soluble in water.
A part of the water soluble proteid matter obtained as above is
coagulated by heat at from 50° to 80°. The part not separated by heat
gives a precipitate on saturation with sodium chlorid.
In wheat there are found soluble in water two albumins and a
proteose.[371]
In separating the albumin coagulating at a low boiling point from the
dialyzed solution mentioned above, it is heated to 60° for an hour,
the precipitate collected on a gooch, washed with hot water (60°), and
then successively with ninety-five per cent alcohol, water-free alcohol
and ether. On drying the residual voluminous matter on the filter over
sulfuric acid, it becomes dense and horny, having in an ash free state,
according to Osborne, the following composition:
Per cent.
Carbon 53.06
Hydrogen 6.82
Nitrogen 17.01
Sulfur 1.30
Oxygen 21.81
=398. Treatment without Precipitation with Ammonium Sulfate.=—Where
abundant means are at hand for dialyzing large volumes of solution, the
preliminary treatment of the solution made with ten per cent sodium
chlorid with ammonium sulfate may be omitted.
When the precipitated proteids are to be used for the estimation of
the nitrogen therein contained, it has been proposed to substitute
the corresponding zinc salt for the ammonium sulfate.[372] This
reagent has given satisfactory results in this laboratory and while a
larger experience is desirable before commending it as an acceptable
substitute in all cases, yet its obvious advantage, in being free of
nitrogen for the use mentioned, entitles it to careful consideration.
The manipulation, with the exception of the precipitation with ammonium
sulfate, is the same as that described in the preceding paragraph. The
globulins are completely precipitated when the dialysis is complete and
may be separated from the soluble albumins and proteoses by filtration.
=399. Separation of the Bodies Soluble in Water.=—_Albumins._—By the
methods of treatment just described, the proteid matters soluble in
ten per cent sodium chlorid solution are separated into two classes,
_viz._, globulins insoluble in pure water and albumins and proteoses
soluble in pure water. The aqueous solution will also contain any amids
or nitrogenous bases soluble in the dilute saline solution and in
water. Osborne and Voorhees have found that the best way of separating
the albumins in the pure aqueous solution is by the application of
heat.[373] By means of a fractional coagulation the albumins are
divided into classes, _viz._, those separating at from 60° to 65° and
those remaining in solution at that temperature but separating up to
85°. The respective quantities of these albumins are determined by
collecting them in a filter and estimating the nitrogen therein by
moist combustion in the usual way. Even a larger number of albumins may
be secured, as in the maize kernel, by such a fractional precipitation
by means of heat. Chittenden and Osborne find in this instance that the
precipitation begins at about 40°.[374]
_Proteose._—After the separation of the albumins by heat the filtrate
may still contain proteid matter. This matter belongs to the proteose
class. It may be partially secured by concentrating the filtrate,
after the removal of the albumins, to a small bulk when a part of
the proteose body will separate. It may be thrown out entirely by
treating the filtrate above mentioned with fine-ground salt until it is
saturated or by adding salt until the solution contains about twenty
per cent thereof and precipitating the proteose by acetic acid.[375]
=400. Separation of the Globulins.=—The globulins which are extracted
with ten per cent solution of sodium chlorid and precipitated on
dialysis may be separated by fractional solution into several bodies of
nearly related properties. This solution is conveniently accomplished
by saline solvents of increasing strength. In the case of the maize
globulins, Chittenden and Osborne employ dilute solutions of common
salt for effecting the separation, beginning with a quarter of a per
cent and ending with a two per cent mixture.[376]
=401. Proteids Soluble in Dilute Alcohol.=—Some of the proteid bodies
which are soluble in dilute salt solution and in water are also soluble
in alcohol. Since these bodies are more easily identified by the
processes already described, attention will be given in this paragraph
solely to those proteid bodies which are insoluble in water or dilute
salt solution and are soluble in dilute alcohol.
For the extraction of these bodies, the residue, left after extraction
with a ten per cent solution of sodium chlorid or with water, is
mixed with enough strong alcohol to secure by the admixture with the
water present in the sample an alcohol of about seventy-five per cent
strength. The mixture is well shaken and digested for some time, at a
temperature of about 46°, and thrown on a filter which is kept at about
the same temperature. The residue is again mixed with alcohol of the
same strength (seventy-five per cent) using about four liters for two
and a half kilos of the original material. During the second digestion
the temperature is kept at about 60°. The latter operation is repeated
three times and in each case the filtrate obtained is evaporated
separately.[377] This process is especially applicable to the meal from
maize kernels, which contains a high relative percentage of an alcohol
soluble proteid, zein.
The chief part of the zein is found in the first two extracts, obtained
as described above. On evaporation, the zein separates as a tough,
leathery, yellow-colored mass on the walls of the containing vessel. It
is cut into small pieces and digested for several days in cold, pure
alcohol. This is followed by digestion with a mixture of ether and
pure alcohol, and finally with pure ether. By this treatment a part of
the zein becomes insoluble in seventy-five per cent alcohol. The part
soluble in dilute alcohol is precipitated by pouring it into water.
Another method of preparing zein is to extract the meal with
seventy-five per cent alcohol after it has been treated with a ten per
cent salt solution.
In this case the extraction is continued with seventy-five per cent
alcohol in successive portions until no more proteid matter passes
into solution. The several extracts are united and the alcohol removed
by distillation, by which process the zein is separated. It is washed
with distilled water, until the sodium chlorid is removed, dissolved
in warm alcohol of about eighty per cent strength and any insoluble
matter removed by filtration. On evaporating the filtrate nearly
to dryness, the zein is separated and pressed as free of water as
possible, yielding a yellow, elastic substance resembling molasses
candy. This preparation is purified by digestion with pure alcohol
and ether in the manner described. The two zeins which are secured
by the treatment, one soluble and the other insoluble in alcohol, are
practically identical in composition.[378]
Zein freshly precipitated by pouring its alcoholic solution in water
is wholly insoluble in water, and, on boiling therewith, is changed
into the variety insoluble in dilute alcohol. Boiled with dilute
sulfuric acid, six in 300 cubic centimeters of water, it melts, forming
a gummy mass, which is very slowly attacked by the acid yielding
proteoses and peptones. Heated with stronger sulfuric acid it undergoes
decomposition, yielding leucin, tyrosin, and glutamic acid.
=402. Solvent Action of Acids and Alkalies.=—In the preceding
paragraphs, a synopsis has been given of the methods of separating
proteid matters in such a manner as to secure them in a pure state in
the same conditions as they exist in the natural substances. A very
large percentage of the proteid matter is still left undissolved after
extraction with the solvents already mentioned.
Often important information may be gained concerning the nature of the
residual proteid matters by fractional extraction with dilute acids
and alkalies. When the strength of these solutions is such that they
contain about one per cent of the acid or alkali, the whole of the
proteid matter may be dissolved by boiling successively with acid and
alkali for half an hour. The proteid matter passing into solution in
these cases is usually changed in character, assuming the nature of
proteoses or allied bodies, when treated with an acid, and becoming
albuminates when boiled with an alkali. Easily soluble carbohydrate
matter is also removed by this treatment so that the residue obtained
consists largely of cellulose and is known as crude or insoluble fiber.
The removal of all the bodies soluble in dilute boiling acid and alkali
is accomplished by the method described in paragraph =272=.
For research purposes, the solvent action of dilute alkali is of chief
importance to the analyst, and the extraction of the proteid matter,
after all that is soluble in water, common salt solution and alcohol
has been removed, should commence with a solution of potassium or
sodium hydroxid containing not over two-tenths per cent of the alkali.
It has been shown by Osborne that the solvent action of very dilute
alkali, in the cold, may be exerted without changing the character of
the dissolved proteid.[379]
=403. Method of Extraction.=—The solvent employed is usually a
two-tenths per cent solution of potassium hydroxid. It may be added
directly to the substance or may follow extraction with water,
salt solution or alcohol. In the former case, the manipulation
is illustrated by the following description of the treatment of
oatmeal:[380]
One hundred grams of oatmeal are mixed with half a liter of a
two-tenths per cent potassium hydroxid solution and allowed to stand
for some time at room temperature. The mixture is strained through
a cloth to remove the chaff and the residue is stirred with another
small portion of the solvent, again strained in the same cloth and the
residue squeezed dry. The strained liquids are united and enough more
of the solvent added to make the volume 700 cubic centimeters. After
standing for some time, the insoluble matter settles to the bottom of
the vessel and the supernatant liquid is decanted. More solvent is
added to the residue, well mixed therewith and treated as above. It is
advisable to make a third extraction in the same way. The extracts are
united, passed through a filter, the proteid matter in solution thrown
out by acetic acid, washed with water, alcohol and ether and dried over
sulfuric acid.
The methods of procedure, when the sample has been previously extracted
with water, salt solution or alcohol, are essentially the same as that
just described and the reader may consult the paper of Osborne for
details.[381]
=404. Methods of Drying Separated Proteids.=—In the preceding
paragraphs, the analyst has been directed, in most instances, to
dry the proteid matter, after it is secured in as pure a form as
possible, at room temperature, over sulfuric acid. By this treatment
the preparation may be obtained in a form suited to the study of its
physical properties, since its solubility has not been affected by
subjecting it to a high temperature. When it is desired to use the
sample only for chemical analysis it is not necessary to wait on the
slow process above mentioned. In this case the sample may be dried in
an inert atmosphere at the temperature of a steam-bath or even at 110°.
It is better, however, to avoid so high a temperature and to conduct
the desiccation in vacuo at a heat not above that of boiling water. The
sample, before drying, should be reduced to the finest possible state
of comminution, otherwise particles of aqueous vapor may be retained
with great tenacity.
In many cases it is advisable to dry the sample pretty thoroughly, then
grind to a fine powder and finish the desiccation with the pulverulent
mass. This treatment can be followed when the quantity of the material
is considerably in excess of that required for the analytical
operations.
=405. Determination of Ash.=—No method of treatment is known by means
of which vegetable proteid matters may be obtained entirely free
of mineral matters. The mineral bases may be naturally present in
the proteid matter as organic and inorganic salts, or they may be
mechanically entangled therewith, having been derived either from
the other tissues of the plant or from the solvents employed. It is
necessary in calculating the analytical data to base the computation on
the ash free substance. The percentage of ash is determined by any of
the standard processes or by heating the sample in a combustion tube,
to very low redness, in a current of oxygen. The total residue obtained
is used in calculating the percentage of ash, and the weights of
material subsequently used for the determination of carbon, hydrogen,
nitrogen and sulfur are corrected for the calculations by deducting the
quantity of mineral matter contained therein.
By reason of the highly hygroscopic nature of the dry proteid bodies,
they must be kept over a desiccating material and weighed quickly on a
balance, in an atmosphere which is kept free of moisture by the usual
methods.
=406. Carbon and Hydrogen.=—Carbon and hydrogen are estimated in
proteid matters by combustion with copper oxid. Osborne prefers to burn
the sample in a platinum boat in a current of air or of oxygen free of
moisture and carbon dioxid.[382] It is advisable to use also a layer of
lead chromate in addition to the copper oxid and metallic copper. The
method of conducting the combustion has already been described.[383] The
analyst should have at his disposal a quantity of pure sugar, which
may be used from time to time in testing the accuracy of the work.
In beginning a series of combustions this precaution should never be
omitted. The addition of the lead chromate is to make more certain the
absorption of oxidized sulfur produced during the combustion.
=407. Estimation of Nitrogen.=—In most cases it is found convenient,
during the progress of separating vegetable proteids, to determine the
quantity of each kind by estimating the nitrogen by moist combustion
and computing the quantity of proteid matter by multiplying the
nitrogen by 6.25. The estimation of the nitrogen is made either on an
aliquot part of the extract or by direct treatment of the residue.
In the pure extracted proteid matter the nitrogen is most conveniently
determined by moist combustion, but it may also be obtained either by
combustion with soda-lime or with copper oxid, or by other reliable
methods.[384]
The percentages of nitrogen found in the principal proteid bodies,
together with the factors for computing the weights of the proteid
bodies from the weights of nitrogen found, are given below:
Name of body. Percentage of nitrogen. Factor.
Mucin 13.80 to 14.13 7.25 to 7.08
Chondrin 14.20 to 14.65 7.04 to 6.83
Albuminates 13.87 7.21
Oat proteids 15.85 6.31
Serum globulin 15.63 6.40
Egg albumin 15.71 to 17.85 6.37 to 5.60
Maize proteids 16.06 6.22
Casein 15.41 to 16.29 6.49 to 6.13
Serum albumin 15.96 6.27
Syntonin 16.10 6.21
Keratin 16.20 to 17.70 6.17 to 5.65
Fibrinogen 16.65 6.01
Peptones 16.66 to 17.13 6.00 to 5.84
Elastin 16.75 5.97
Wheat proteids 16.80 to 18.39 5.95 to 5.44
Fibrin 16.91 5.91
Flax seed proteids 17.70 to 18.78 5.65 to 5.33
=408. Determination of Sulfur.=—Sulfur is a characteristic constituent
of the proteid bodies, existing in quantities approximating one per
cent of their weight.
In the estimation of sulfur, it is first converted into sulfuric acid,
which is thrown out by a soluble barium salt and the sulfur finally
weighed as barium sulfate.
All the sulfur existing in the organic state in a proteid may be
obtained by burning in a current of oxygen and conducting the gaseous
products of combustion through solid sodium or potassium carbonate
at or near a red heat.[385] The organic sulfur may also be converted
into sulfuric acid by fusing the proteid body with a mixture of
sodium hydroxid and potassium nitrate. The fused mass, after cooling,
is dissolved in water, the solution acidified with hydrochloric,
evaporated to dryness to decompose nitrates and remove excess of
hydrochloric acid and dissolved in a large excess of water. After
standing for a day, the solution is filtered and the sulfuric acid
thrown out of the hot filtrate with a slight excess of barium chlorid
solution. The usual precautions in precipitating, filtering and
igniting the barium sulfate are to be observed.[386]
=409. General Observations.=—In the preceding paragraphs have been
stated the general principles upon which the separation of vegetable
proteid matters depends, and a description has been given of the
several processes by which this separation is accomplished. In
each case, however, special conditions exist which require special
modifications of the general processes, and these can only be
successfully secured by the skill, judgment and patient labor of the
investigator. Many of these cases have been already worked out, and the
valuable data secured by Chittenden, Osborne and others, are accessible
to the analyst in the papers already cited. In the case of the proteids
in the peanut, a similar work has been done in this laboratory by
Bigelow, the data of which have not yet been published. It is only
by a careful study of the work already done as outlined here and as
published in full in the cited papers, that the analyst will be able to
secure trustworthy guidance for future investigations.
=410. Dialysis.=—One of the most important of the operations connected
with the separation and analysis of proteids is the removal of the
salts whereby their solutions are secured. This is accomplished by
subjecting the solutions of the proteid matters to dialysis. The
solution is placed in bags made of parchment dialysis paper. These
bags are tied about a glass tube, whereby access may be had to their
contents during the progress of the work. Since the volume of the
liquid increases during the process, the bags should not be filled too
full in the beginning.
[Illustration: FIG. 105. DIALYZING APPARATUS.]
In this laboratory the dialysis is carried out by Bigelow with the
city water from the Potomac, which is first passed through a battery
of porous porcelain filtering tubes to remove any suspended silt or
micro-organisms. If unfiltered water be used, the germs therein cause
a fermentation in the proteid matter, which seriously interferes with
the value of the data obtained, and which can only be avoided by the
use of an antiseptic, such as an alcoholic solution of thymol. Even
with filtered water, the use of a few drops of the solution mentioned
is often necessary. To avoid the use of too great quantities of the
filtered water, the dialyzers are arranged _en batterie_, as shown in
the figure. The filtered water enters the first vessel and thence
passes through all. The parchment bags are frequently changed from
vessel to vessel, each being brought successively into the first vessel
in contact with the fresh water. In some cases the final steps in the
dialysis may be accomplished in distilled water.
It is advisable to conduct a fractional preliminary dialysis of the
salt solution containing proteids in such a way as to secure the
globulins precipitated in each interval of twenty-four hours. Each
portion thus secured may be examined with the microscope. Usually a
period of two weeks is required to entirely remove the mineral salts
from solution. If prepared parchment tubes be used for the dialysis,
they should be first tested for leaks, and should not be more than half
filled. By the use of a large number of these tubes a greater surface
is exposed to dialytic action, and the time required to complete the
operation is correspondingly decreased.