Healthy animals in the prime of life are used, and the feeding experiments are conducted with as large a number of animals as possible, in order to eliminate the effects of idiosyncrasy. The food used is previously prepared in abundant quantity and its composition determined by the analysis of an average sample.

The feeding period is divided into two parts. In the first part the animal is fed for a few days with the selected food until it is certain that all the excreta are derived from the nutrients used. In the second part the same food is continued and the excreta collected, weighed, the moisture determined, and the total weight of the water-free excreta ascertained. The first part should be of at least seven and the second of at least five days duration. The urine and dung are analyzed separately. Males are preferred for the digestion experiments because of the greater ease of collecting the urine and feces without mixing. For ordinary purposes the feces only are collected. The methods of analysis do not differ from those described for the determination of the usual ingredients of a food.

Example.—The following data taken from the results of digestive experiments, obtained at the Maine Station, will illustrate the method of comparing the composition of the food with that of the feces and of determining the degree of digestion which the proteids and other constituents of the food have undergone.

Composition of Maize Fodder and of Feces
Therefrom after Feeding to Sheep.

In the above instance it is seen that the coefficient of digestibility extended from 37.0 per cent in the case of the mineral components of the food, to 78.3 per cent in the case of the fats. These data are taken only from the results obtained from a single sheep and one article of food. The mean data secured from two animals and three kinds of maize fodder show the following per cents of digestibility: Ash 39.4, proteid 61.8, fiber 76.7, undetermined matters 72.1, fat 76.4. The undetermined matters are those usually known as nitrogen free extract and composed chiefly of pentosans and other carbohydrates.[569]

554. Natural Digestibility of Pentosans.—The digestibility of pentosan bodies in foods under the influence of natural ferments has been investigated by Lindsey and Holland.[570] The feeding and collection of the feces is carried on as described above and the relative proportions of pentosan bodies in the foods and feces determined by estimating the furfuraldehyd as prescribed in paragraph 150.[571]

PRESERVED MEATS.

555. Methods of Examination.—In general the methods of examination are the same as those applied in the study of fresh meats. The contents of water, salt and other preservatives, fat and nitrogenous matters are of most importance. When not already in a fine state, the preserved meats are run through meat cutters until reduced to a fine pulp. Most potted meats are already in a state of subdivision well suited to analytical work. The composition of preserved meats has been thoroughly studied in this laboratory by Davis.[572]

556. Estimation Of Fat.—Attention has already been called to the difficulty of extracting the fat from meats by ether or other solvents.[573] In preserved meats, as well as in fresh, it is preferable to adopt some method which will permit of the decomposition of the other organic matters and the separation of the fat in a free state. The most promising methods are those employed in milk analyses for the solution of nitrogenous matters. Sulfuric or hydrochloric acid may be used for this purpose, preference being given to sulfuric. The separated fats may be taken up with ether or separated by centrifugal action. A method of this kind for preserved meats, suggested by Hefelmann, is described below.

About six grams of the moist preserved meat are placed in a calibrated test tube and dissolved in twenty-five cubic centimeters of fuming hydrochloric acid. The tube is placed in a water bath, quickly heated to boiling and kept at that temperature for half an hour. About twenty cubic centimeters of cold water are added and the temperature lowered to 30°, then twenty cubic centimeters of ether and the tube gently shaken to promote the solution of the fat. When the ether layer has separated, its volume is read and an aliquot part removed by means of a pipette, dried and weighed. The separation of the ethereal solution is greatly promoted by whirling.

The mean proportions of the ingredients of preserved meats are about as follows:

557. Meat Preservatives.—Various bodies are used to give taste and color to preserved meats and to preserve them from fermentation. The most important of these bodies are common salt, potassium and sodium nitrates, sulfurous, boric, benzoic and salicylic acids, formaldehyd, saccharin and hydronaphthol. A thorough study of the methods of detecting and isolating these bodies has been made in this laboratory by Davis and the results are yet to be published as a part of Bulletin 13.

DETERMINATION OF NUTRITIVE VALUES.

558. Nutritive Value of Foods.—The value of a food as a nutrient depends on the amount of heat it gives on combustion in the tissues of the body, i. e. oxidation, and in its fitness to nourish the tissues of the body, to promote growth and repair waste. The foods which supply heat to the body are organic in their nature and are typically represented by fats and carbohydrates. The foods which promote growth and supply waste are not only those which preeminently supply heat, but also include the inorganic bodies and organic nitrogenous matters represented typically by the proteids. It is not proper to say that one class of food is definitely devoted to heat forming and another to tissue building, inasmuch as the same substance may play an important rôle in both directions. As heat formers, carbohydrates and proteids have an almost equal value, as measured by combustion in oxygen, while fat has a double value for this purpose. The assumption that combustion in oxygen forms a just criterion for determining the value of a food must not be taken too literally. There are only a few bodies of the vast number which burn in oxygen that are capable of assimilation and oxidation by the animal organism. Only those parts of the food that become soluble and assimilable under the action of the digestive ferments, take part in nutrition and the percentage of food materials digested varies within wide limits but rarely approaches 100. It may be safely said that less than two-thirds of the total food materials ingested are dissolved, absorbed, decomposed and assimilated in the animal system. We have no means of knowing how far the decomposition (oxidation) extends before assimilation, and therefore no theoretical means of calculating the quantity of heat which is produced during the progress of digestion. The vital thermostat is far more delicate than any mechanical contrivance for regulating temperature and the quantity of food, in a state of health, converted into heat, is just sufficient to maintain the temperature of the body at a normal degree. Any excess of heat produced, as by violent muscular exertion, is dissipated through the lungs, the perspiration and other secretions of the body.

Pure cellulose or undigestible fiber, when burned in oxygen, will give a thermal value approximating that of sugar, but no illustration is required to show that when taken into the system the bodily heat afforded by it is insignificant in quantity.

Thermal values, therefore, have little comparative usefulness in determining nutritive worth, except when applied to foods of approximately the same digestive coefficient.

559. Comparative Value of Food Constituents.—It has already been noted that, judged by combustion in oxygen, carbohydrates and proteids have about half the thermal value possessed by fats. Commercially, the values of foods depend in a far greater degree on their flavor and cooking qualities than upon the amount of nutrition they contain. Butter fat, which is scarcely more nutritious than tallow, is worth twice as much in the market, while the prices paid for vegetables and fruits are not based to any great extent on their food properties.[574] In cereals, especially in wheat, the quantity of fat is relatively small, and starch is the preponderating element. In meats, carbohydrates are practically eliminated and fats and proteids are the predominating constituents.

In the markets, fats and proteids command far higher prices than sugars and starches. The relative commercial food value of a cereal may be roughly approximated by multiplying the percentages of fat and protein by two and a half and adding the products to the percentage of carbohydrates less insoluble fiber. This method was adopted in valuing the cereals at the World’s Columbian Exposition.[575]

560. Nutritive Ratio.—In solid foods the nutritive ratio is that existing between the percentage of proteids and that of carbohydrates, increased by multiplying the fat by two and a half and adding the product. In a cereal containing twelve per cent of protein, seventy-two of carbohydrates, exclusive of fiber, and three of fat, the ratio is 12: 72 + 3 × 2.5 = 6.5. Instead of calculating the nutritive ratio directly from the data obtained by analysis, it may be reckoned from the per cents of the three substances in the sample multiplied by their digestive coefficient. Since the relative amounts of proteids, fats and carbohydrates digested do not greatly differ, the numerical expression of the nutritive ratio is nearly the same when obtained by each of these methods of calculation.

Where the proportion of protein is relatively large the ratio is called narrow, 1: 4 ... 6. When the proportion of protein is relatively small the ratio is called broad 1: 8 ... 12. In feeding, the nutritive ratio is varied in harmony with the purpose in view, a narrow ratio favoring the development of muscular energy, and a wide one promoting the deposition of fat and the development of heat. These principles guide the scientific farmer in mixing rations for his stock, the work horses receiving a comparatively narrow and the beeves a relatively wide ratio in their food.

561. Calorimetric Analyses of Foods.—The general principles of calorimetry have been already noticed. The theoretical and chemical relations of calorimetry have been fully discussed by Berthelot, Thomsen, Ostwald and Muir.[576] In the analyses of foods the values as determined by calculation or combustion are of importance in determining the nutritive relations.

Atwater has presented a résumé of the history and importance of the calorimetric investigations of foods to which the analyst is referred.[577]

In the computation of food values the percentages of proteids, carbohydrates and fats are determined and the required data obtained by applying the factors 4100, 5500 and 9300 calories for one gram of carbohydrates, proteids and fats respectively.

For most purposes the computed values are sufficient, but it is well to check them from time to time by actual combustions in a calorimeter.

562. Combustion in Oxygen.—The author made a series of combustions of carbonaceous materials in oxygen at the laboratory of Purdue University in 1877, the ignition being secured by a platinum wire rendered incandescent by the electric current. The data obtained were unsatisfactory on account of the crudeness of the apparatus. The discovery of the process of burning the samples in oxygen at a high pressure has made it possible to get expressions of thermal data which while not yet perfect, possess a working degree of accuracy. The best form of bomb calorimeter heretofore employed is that of Hempel, as modified by Atwater and Woods.[578]

A section of this calorimeter, with all the parts in place, is shown in Fig. 120.

In the figure the steel cylinder A, about 12.5 centimeters deep and 6.2 in diameter, represents the chamber in which the combustion takes place. Its walls are about half a centimeter thick and it weighs about three kilograms. It is closed, when all the parts are ready and the sample in place, by the collar C, which is secured gas tight by means of a powerful spanner. The cover is provided with a neck D carrying a screw E and a valve screw F. In the neck D, where the bottom of the cylinder screw E rests, is a shoulder fitted with a lead washer. Through G the oxygen used for combustion is introduced. The upper edge of the cylinder A is beveled and fits into a groove in the cover B, carrying a soft metal washer. To facilitate the screwing on of the cover, ball bearings KK, made of hard steel, are introduced between the collar and the cover. The platinum wires H and I support the platinum crucible holding the combustible bodies which are ignited by raising the spiral iron wire connecting them to the temperature of fusion by an electric current. The combustion apparatus when charged is immersed in a metal cylinder M, containing water and resting on small cylinders of cork. The water is stirred by the apparatus LL. The cylinder M is contained in two large concentric cylinders, N, O, made of non-conducting materials and covered with disks of hard rubber. The space between O and N may be filled with water. The temperature is measured by the thermometer P, graduated to hundredths of a degree and the reading is best accomplished by means of a cathetometer.

563. The Williams Calorimeter.—The calorimeter bomb has been improved by Williams by making it of aluminum bronze of a spheroidal shape. The interior of the bomb is plated with gold. By an ingenious arrangement of contacts the firing is secured by means of a permanently insulated electrode fixed in the side of the bomb. The calorimetric water, as well as that in the insulating vessel, is stirred by means of an electrical screw so regulated as to produce no appreciable degree of heat mechanically. The combustion is started by fusing a fine platinum wire of definite length and thickness by means of an electric current. The heat value of this fusion is determined and the calories produced deducted from the total calories of the combustion. The valve admitting the oxygen is sealed automatically on breaking connection with the oxygen cylinder. The effluent gases, at the end of the combustion, may be withdrawn through an alkaline solution and any nitric acid therein thus be fixed and determined.[579]

564. Manipulation and Calculation.—The material to be burned is conveniently prepared by pressing it into tablets. The oxygen is supplied from cylinders, of which two should be used, one at a pressure of more than twenty atmospheres. By this arrangement a pump is not required.

In practical use, a known weight of the substance to be burned is placed in the platinum capsule, the cover of the bomb screwed on, after all adjustments have been made, and the apparatus immersed in the water contained in M, which should be about 2° below room temperature. All the covers are placed in position and the temperature, of the water in M begins to rise. Readings of the thermometer are taken at intervals of about one minute for six minutes, at which time the temperature of the bomb and calorimetric water may be regarded as sensibly the same. The electric current is turned on, the iron wire at once melts, ignites the substance and the combustion rapidly takes place. In the case of bodies which do not burn readily Atwater adds to them some naphthalene, the thermal value of which is previously determined. The calories due to the combustion of the added naphthalene are deducted from the total calories obtained.

The temperature of the water in M rises rapidly at first, and readings are made at intervals of one minute for five minutes, and then again after ten minutes. The first of the initial readings, the one at the moment of turning on the current, and the last one mentioned above are the data from which the correction, made necessary by the influence of the temperature of the room, is calculated by the following formulas.[580]

The preliminary readings of the thermometer at one minute intervals are represented by t₁, t₂, t₃ ... tₙ₁. The last observation tₙ₁ is taken as the beginning temperature of the combustion and is represented in the formulas for calculations by Θ₁. The readings after combustion are also made at intervals of one minute, and are designated by Θ₂, Θ₃ ... Θₙ. The readings are continued until there is no observed change between the last two. Generally this is secured by five or six readings.

The third period of observations begins with the last reading Θₙ, which in the next series is represented by tʹ₁, tʹ₂ ... tʹₙ₂.

In order to make the formulas less cumbersome let

The correction to be made to the difference between Θₙ - Θ₁ for the influence of the outside temperature is determined by the formula of Regnault-Pfaundler, which is as follows:

is calculated from the observation of the thermometer Θ₁, Θ₂ etc., made immediately after the combustion. It is equal to the sum of observations Θ₁, Θ₂ etc., increased by an arbitrary factor equivalent to (Θ₂ - Θ₁)/9, which is made necessary by reason of the irregularity of the temperature increase during the first minute after combustion, the mean temperature during that minute being somewhat higher than the mean of the temperatures at the commencement and end of that time.

The quantity of heat formed by the combustion of the iron wire used for igniting the sample is to be deducted from the total heat produced. This correction may be determined once for all, the weight of the iron wire used being noted and that of any unburned portion being ascertained after the combustion.

Ten milligrams of iron, on complete combustion, will give sixteen calories.

In the combustion of substances containing nitrogen, or in case the free nitrogen of the air be not wholly expelled from the apparatus before the burning, nitric acid is formed which is dissolved by the water produced.

The heat produced by the solution of nitric acid in water is 14.3 calories per gram molecule. The quantity of nitric acid formed is determined by titration and a corresponding reduction made in the total calculated calories.

In the titration of nitric acid it is advisable to make use of an alkaline solution, of which one liter is equivalent to 4.406 grams of nitric acid. One cubic centimeter of the reagent is equivalent to a quantity of nitric acid represented by one calorie.

Since the materials of which the bomb is composed have a specific heat different from that of water, it is necessary to compute the water thermal value of each apparatus.

The hydrothermal equivalent of the whole apparatus is most simply determined by immersing it at a given temperature in water of a different temperature.[581] With small apparatus this method is quite sufficient, but there are many difficulties attending its application to large systems weighing several kilograms. In these cases the hydrothermal equivalent may be calculated from the specific heats of the various components of the apparatus.

In calculating these values the specific heats of the various components of the apparatus are as follows:

Example.—It is required to calculate the hydrothermal value of a calorimeter composed of the following substances:

When a bomb of 300 cubic centimeters capacity is filled with oxygen at a pressure of twenty-four atmospheres it will hold about ten grams of the gas, equivalent to a water value of 2.40 grams. Hence the water value of the above system when charged, assuming the bomb to be of the capacity mentioned, is 380.12 grams.

If the cylinder holding the water be made of fiber or other non-conducting substance, its specific heat is best determined by filling it in a known temperature with water at a definite different temperature.

It is advisable to have the water cylinder of such a size as to permit the use of a quantity of water for the total immersion of the bomb which will weigh, with the water value of the apparatus, an even number of grams. In the case above, 2622.28 grams of water placed in the cylinder will make a water value of 3,000 grams, which is one quite convenient for calculation.

565. Computing the Calories of Combustion.—In the preceding paragraph has been given a brief account of the construction of the calorimeter and of the methods of standardizing it and securing the necessary corrections in the data directly obtained in its use. An illustration of the details of computing the calories of combustion taken from the paper of Stohmann, Kleber and Langbein, will be a sufficient guide for the analyst in conducting the combustion and in the use of the data obtained.[582]

The thermometric readings are given in the divisions of the thermometer which in this case are so adjusted as to have the number 28.90 correspond to 12°.8406, and each division is nearly equivalent to 0°.014 thermometric degree.

The number of calories above given is the proper one when the computation is made to refer to constant volume. By reason of the consumption of oxygen and the change of temperature, although mutually compensatory, the pressure may be changed at the end of the operation. The conversion of the data obtained at constant volume referred to constant pressure may be made by the following formula, in which [Q] represents the calories from constant volume and Q the desired data for constant pressure, O the number of oxygen atoms, H the number of hydrogen atoms in a molecule of the substance, and 0.291 a constant for a temperature of about 18°, at which the observations should be made.

566. Calorimetric Equivalents.—By the term calorie is understood the quantity of heat required to raise one gram of water, at an initial temperature of about 18°, one degree. The term ‘Calorie’ denotes the quantity of heat, in like conditions, required to raise one kilogram of water one degree.

For purposes of comparison and for assisting the analyst in adjusting his apparatus so as to give reliable results, the following data, giving the calories of some common food materials, are given:

567. Distinction between Butter and Oleomargarin.—Theoretically the heats of combustion of butter fat and oleomargarin are different and de Schweinitz and Emery propose to utilize this difference for analytical purposes.[583] The samples of pure butter fat examined by them afforded 9320, 9327 and 9362 calories, respectively. The calories obtained for various samples of oleomargarin varied from 9574 to 9795. On mixing butter fat and oleomargarin, a progressive increase in calorimetric power is found, corresponding to the percentage of the latter constituent. Lards examined at the same time gave from 9503 to 9654 calories.

FRUITS, MELONS AND VEGETABLES.

568. Preparation of Sample.—Fresh fruits and vegetables are most easily prepared for analysis by passing them through the pulping machine described on page 9. Preliminary to the pulping they should be separated into skins, cores, seeds and edible portions, and the respective weights of these bodies noted. Each part is separately reduced to a pulp and, at once, a small quantity of the well mixed substance placed in a flat bottom dish and dried, first at a low temperature, and finally at 100°, or somewhat higher, and the percentage of water contained in the sample determined. The bulk of the sample, three or four kilograms, is dried on a tray of tinned or aluminum wire, first at a low and then at a high temperature, until all or nearly all the moisture is driven off. The dried pulp is then ground to as fine a powder as possible and subjected to the ordinary processes of analysis; viz., the determination of the moisture, ash, nitrogen, fiber, fat and carbohydrates.

In this method of analysis it is customary to determine the carbohydrates, exclusive of fiber, by subtracting the sum of the per cents of the other constituents and the nitrogen multiplied by 6.25 from 100.

569. Separation of the Carbohydrates.—It is often desirable to determine the relative proportions of the more important carbohydrates which are found in fruits and vegetables. The pentoses and pentosans are estimated by the method described in paragraph 150. The cane sugar, dextrose and levulose are determined by extracting a portion of the substance with eighty per cent alcohol and estimating the reducing sugars in the extract before and after inversion by the processes described in paragraphs 238-251. The percentages of sugars deducted from the percentage of carbohydrates, exclusive of fiber, give the quantity of gums, pentosans, cellulose and pectose bodies present.

Pectose exists chiefly in unripe fruits. By the action of the fruit acids and of a ferment, pectose, in the process of ripening, is changed into pectin and similar hydrolyzed bodies soluble in water. The gelatinous properties of boiled fruits and fruit juices are due to these bodies, boiling accelerating their formation. In very ripe fruits the pectin is completely transformed into pectic acids. The galactan is estimated as described in 585.

570. Examination of the Fresh Matter.—To avoid the changes which take place in drying fruits and vegetables, it is necessary to examine them in the fresh state. The samples may be first separated into meat and waste, as suggested above, or shredded as a whole. The moisture in the pulp is determined as indicated above, and in a separate portion the soluble matters are extracted by repeated treatment with cold water. The seeds, skins, cellulose, pectose and other insoluble bodies are thus separated from the sugars, pectins, pectic and other acids, and other soluble matters. The insoluble residue is rapidly dried and the relative proportions of soluble and insoluble matters determined. The estimation of these bodies is accomplished in the usual way.

571. Examination of Fruit and Vegetable Juices.—The fruits and vegetables are pulped, placed in a press and the juices extracted. The pressure should be as strong as possible and the press described in paragraph 280 is well suited to this purpose. The specific gravity of the expressed juice is obtained and the sucrose therein determined by polarization before and after inversion. The reducing sugars and the relative proportions of dextrose and levulose are determined in the usual manner. In grape juice dextrose is the predominant sugar while in many other fruits left hand or optically inactive sugars predominate. Soluble gums, dextrin, pectin etc., may be separated from the sugars by careful precipitation with alcohol, or the total solids, ash, nitrogen, ether extract and acids be determined and the carbohydrates estimated by difference. From the carbohydrates the total percentage of sugars is deducted and the remainder represents the quantity of pectin, gum and other carbohydrates present.

572. Separation of Pectin.—Pectin exists in considerable quantities in the juice of ripe fruits (pears) and may be obtained in an approximately pure state from the juices by first removing proteids by the careful addition of tannin, throwing out the soluble lime salts with oxalic acid and precipitating the pectin with alcohol. On boiling with water, pectin is converted into parapectin, which gives a precipitate with lead acetate. Boiling with dilute acids converts pectin into metapectin, which is precipitated by a barium salt.

Pectic acid may be obtained by boiling an aqueous extract (carrots) with sodium carbonate and precipitating the pectic with hydrochloric acid. It is a jelly-like body and dries to a horny mass.

573. Determination of Free Acid.—The free acid, or rather total acidity of fruits, is determined by the titration of their aqueous extracts or expressed juices with a set alkali. In common fruits and vegetables the acidity is calculated to malic C₄H₆O₅, in grapes to tartaric C₄H₆O₆, and in citrous fruits to citric acid C₆H₈O₇. Many other acids are found in fruits and vegetables, but those mentioned are predominant in the classes given.

574. Composition of Common Fruits.—The composition of common fruits in this country has been extensively investigated at the California Station and the following data are derived chiefly from its bulletins.[584]

• (A) = Total sugars in juice.
• (B) = Sucrose in juice.
• (C) = Dry organic matter.

575. Composition of Ash of Fruits.—Two or three kilograms of the dried sample are incinerated at a low temperature and burned to a white ash in accordance with the directions given in paragraphs 28-32.

The composition of the ash is determined by the methods already described.[586]

The pure ash of some common whole fruits has the following composition:[587]

• (A) = Per cent pure ash in fruit.
• (B) = Per cent manganomanganic oxid.
• (C) = Per cent phosphorus pentoxid.
• (D) = Per cent sulfur trioxid.

576. Dried Fruits.—A method of preserving fruits largely practiced consists in subjecting them, in thin slices or whole, to the action of hot air until the greater part of the moisture is driven off. The technique of the process is fully described in recent publications.[588] It has been shown by Richards that fruit subjected to rapid evaporation undergoes but little change aside from the loss of water.[589]

In the analyses of dried fruits the methods already described are used. The presence of pectin renders the filtration of the aqueous extract somewhat difficult, and in many cases it is advisable to determine the sugars present in the extract without previous filtration.

577. Zinc in Evaporated Fruits.—Fruits are commonly evaporated on trays made of galvanized iron. In these instances a portion of the zinc is dissolved by the fruit acids, and will be found as zinc malate etc., in the finished product. The presence of zinc salts is objectionable for hygienic reasons, and therefore the employment of galvanized trays should be discontinued. The presence of zinc in evaporated fruits may be detected by the following process.[590] The sample is placed in a large platinum dish and heated slowly until dry and in incipient combustion. The flame is removed and the combustion allowed to proceed, the lamp being applied from time to time in case the burning ceases. When the mass is burned out it will be found to consist of ash and char, which are ground to a fine powder and extracted with hydrochloric or nitric acid. The residual char is burned to a white ash at a low temperature, the ash extracted with acid, the soluble portion added to the first extract and the whole filtered. The iron in the filtrate is oxidized by boiling with bromin water and the boiling continued until the excess of bromin is removed. A drop of methyl orange is placed in the liquid and ammonia added until it is only faintly acid. The iron is precipitated by adding fifty cubic centimeters of a solution containing 250 grams of ammonium acetate in a liter and raising the temperature to about 80°. The precipitate is separated by filtration and washed with water at 80° until free of chlorids. The filtrate is saturated with hydrogen sulfid, allowed to stand until the zinc sulfid settles and poured on a close filter. It is often necessary to return the filtrate several times before it becomes limpid. The collected precipitate is washed with a saturated solution of hydrogen sulfid containing a little acetic acid. The precipitate and filter are transferred to a crucible, dried, ignited and the zinc weighed as oxid. Small quantities of zinc salts added to fresh apples which were dried and treated as above described, were recovered by this method without loss. Other methods of estimating zinc in dried fruits are given in the bulletin cited.

Evaporated apples contain a mean content of 23.85 per cent of water and 0.931 per cent of ash.

The mean quantity of zinc oxid found in samples of apples dried in the United States is ten milligrams for each 100 grams of the fruit, an amount entirely too small to produce any toxic effects. When zinc exists in the soil it will be found as a natural constituent in the crop.[591]

578. Composition of Watermelons and Muskmelons.—In the examination of melons a separation of the rind, seeds and meat is somewhat difficult of accomplishment, since the line of demarcation is not distinct. In watermelons the separation of rind and meat is made at the point where the red color of the meat disappears. In muskmelons no such definite point is found and in the examination of these they are taken as a whole. The total moisture, ash and nitrogen may be determined in the whole mass or in the separate portions. The sugars are most conveniently determined in the expressed juices. The mean composition of the melons given below is that obtained from analyses made in the Department of Agriculture.[592]

TEA AND COFFEE.

579. Special Analysis.—Aside from the examination of teas and coffees for adulterants, the only special determinations which are required in analyses are the estimation of the alkaloid (caffein) and of the tannin contained therein. It is chiefly to the alkaloid that the stimulating effects of the beverages made from tea and coffee are due. The determination of the quantity of tannin contained in tea and coffee is accomplished by the processes described under the chapter devoted to that glucosid.

The general analysis, viz., the estimation of water, ether extract, total nitrogen, fiber, carbohydrates and ash, with the exceptions noted above, is conducted by the methods which have already been given.

For detailed instructions concerning the detection of adulterants of tea and coffee the bulletins of the Chemical Division, Department of Agriculture, may be consulted.[593]

580. Estimation of Caffein (Thein).—The method adopted by Spencer, after a thorough trial of all the usual processes for estimating this alkaloid, is as follows:[594] To three grams of the finely powdered tea or coffee, in a 300 cubic centimeter flask, add about a quarter of a liter of water, slowly heat to the boiling point, using a fragment of tallow to prevent frothing, and boil gently for half an hour. When boiling begins, the flask should be nearly filled with hot water and more added from time to time to compensate for the loss due to evaporation. After cooling, add a strong solution of basic lead acetate until no further precipitation is produced, complete the volume to the mark with water, mix and throw on a filter. Precipitate the lead from the filtrate by hydrogen sulfid and filter. Boil a measured volume of this filtrate to expel the excess of hydrogen sulfid, cool and add sufficient water to compensate for the evaporation. Transfer fifty cubic centimeters of this solution to a separatory funnel and shake seven times with chloroform. Collect the chloroform solution in a tared flask and remove the solvent by gentle distillation. A safety bulb, such as is used in the kjeldahl nitrogen method, should be employed to prevent entrainment of caffein with the chloroform vapors.

The extraction with chloroform is nearly complete after shaking out four times; a delicate test, however, will usually reveal the presence of caffein in the watery residue even after five or six extractions, hence seven extractions are recommended for precautionary reasons. The residual caffein is dried at 75° for two hours and weighed.

The principal objection which has been made to Spencer’s method is that the boiling with water is not continued for a sufficient length of time. For the water extraction, Allen prescribes at least six hours cohobation.[595] In this method six grams of the powdered substance are boiled with half a liter of water for six hours in a flask, with a condenser, the decoction filtered, the volume of the filtrate completed to 600 cubic centimeters with the wash water, heated to boiling, and four cubic centimeters of strong lead acetate solution added, the mixture boiled for ten minutes, filtered and half a liter of the filtrate evaporated to fifty cubic centimeters. The excess of lead is removed with sodium phosphate and the filtrate and washings concentrated to about forty cubic centimeters. The caffein is removed by shaking four times with chloroform. Older but less desirable processes are fully described by Allen.[596]

In France this method is known as the process of Petit and Legrip, and it has been worked out in great detail by Grandval and Lajoux and by Petit and Terbat.[597]

581. Estimation of Caffein by Precipitation with Iodin.—The caffein in this method is extracted, the extract clarified by lead acetate and the excess of lead removed as in Spencer’s process described above. The caffein is determined in the acidified aqueous solution thus prepared, according to the plan proposed by Gomberg, as follows:[598]

Definite volumes of the aqueous solution of the caffein are acidulated with sulfuric and the alkaloid precipitated by an excess of a set solution of iodin in potassium iodid. After filtering, the excess of iodin in an aliquot part of the filtrate is determined by titration with a tenth normal solution of sodium thiosulfate. The filtration of the iodin liquor is accomplished on glass wool or asbestos. The results of the analyses are calculated from the composition of the precipitated caffein periodid; viz., C₈H₁₀N₄O₂.HI.I₄. The weight of the alkaloid is calculated from the amount of iodin required for the precipitation by the equation 4I: C₈H₁₀N₄O₂ = 508: 194. From this equation it is shown that one part of iodin is equivalent to 0.3819 part of caffein, or one cubic centimeter of tenth normal iodin solution is equal to 0.00485 gram of iodin.

In practice, it is recommended to divide the aqueous extract of the alkaloid, prepared as directed above, into two portions, one of which is treated with the iodin reagent without further preparation, and the other after acidulation with sulfuric. After ten minutes, the residual iodin is estimated in each of the solutions as indicated above. The one portion, containing only the acetic acid resulting from the decomposition of the lead acetate, serves to indicate whether the aqueous solution of the caffein contains other bodies than that alkaloid capable of forming a precipitate with the reagent, since the caffein itself is not precipitated even in presence of strong acetic acid.

In the solution acidulated with sulfuric, the caffein, together with the other bodies capable of combining with iodin, is precipitated. The residual iodin is determined in each case, and thus the quantity which is united with the caffein is easily ascertained. The weight of iodin which has entered into the precipitated caffein periodid multiplied by 0.3819 gives the weight of the caffein in the solution.

Gomberg’s method has been subjected to a careful comparative study by Spencer and has been much improved by him in important particulars.[599]

It is especially necessary to secure the complete expulsion of the hydrogen sulfid and to observe certain precautions in the addition of the iodin reagent. The precipitation should be made in a glass-stoppered flask, shaking thoroughly after the addition of the iodin and collecting the precipitate on a gooch. As thus modified, the iodin process gives results comparable with those obtained by Spencer’s method, and it can also be used to advantage in estimating caffein in headache tablets in the presence of acetanilid.

582. Freeing Caffein of Chlorophyll.—Any chlorophyll which may pass into solution and be found in the caffein may be removed by dissolving the caffein in ten per cent sulfuric acid, filtering, neutralizing with ammonia and evaporating to dryness. The residue is taken up with chloroform, the chloroform removed at a low temperature and the pure caffein thus obtained.[600]

583. Proteid Nitrogen.—The proteid nitrogen in tea and coffee may be determined in the residue after extraction of the alkaloid by boiling water as described above. More easily it is secured by determining the total nitrogen in the sample and deducting therefrom the nitrogen present as caffein. The remainder, multiplied by 6.25, will give the quantity of proteid matter.

584. Carbohydrates of the Coffee Bean.—The carbohydrates of the coffee bean include those common to vegetable substances; viz., cellulose, pentosan bodies (xylan, araban), fiber etc., together with certain sugars, of which sucrose is pointed out by Ewell as the chief.[601] In smaller quantities are found a galactose yielding body (galactan), as pointed out by Maxwell, a dextrinoid and a trace of a sugar reducing alkaline copper solution.

The sucrose may be separated from the coffee bean by the following process:[602] The finely ground flour is extracted with seventy per cent alcohol, the extract clarified with lead acetate, filtered, the lead removed from the filtrate with hydrogen sulfid, the excess of the gas removed by boiling, the filtrate evaporated in a partial vacuum to a sirup and the sucrose crystallized from a solution of the sirup in alcohol.

For a quantitive determination, ten grams of the coffee flour are extracted with ether and the residue with seventy-five per cent alcohol. This process, conducted in a continuous extraction apparatus, should be continued for at least twenty-four hours. The alcohol is removed by evaporation, the residue dissolved in water, clarified with basic lead acetate, filtered, the precipitate washed, the lead removed, again filtered, the filtrate washed and wash water and filtrate made to a definite volume. In an aliquot part of this solution the sugars are determined by the alkaline copper method, both before and after inversion. From the data obtained the percentage of sucrose is calculated.