Clarence P . Harris
522 Fif t h Av e., Ne w Yo r k, N . Y .
T
HE well known, if not classical, method for the determination of total fat described by Lewkowitsch (18) consists of mixing the material with four to six times its weight of either sand or finely powdered gypsum, in the case of hygroscopic substances, drying the treated sample at 100° C., and extracting with a suitable solvent in a Soxhlet apparatus. Petroleum ether he considers advisable as sol
vent “ for strictly accurate results,” as this liquid does not dissolve the oxidized acids of sulfur, olive oil, dégras, etc., nor theobromine occurring in cacao beans. Ethyl ether readily extracts this alkaloid (3, 8), but is likely to extract animal fats incompletely owing to the presence of lecithin (11).
Many variations and improvements of this general method have been developed, all of them based upon the principle of isolating and weighing the actual fat present.
This principle is quite sound with materials which have been so treated that the oil or fat is “ free”— that is, not oc
cluded by vegetable or animal tissues. Finely ground cacao nibs (chocolate liquor) or cottonseed press cake (cottonseed meal) are completely extracted without difficulty, but sub
stances like chocolate expeller cake and either raw cotton
seed or cottonseed meats are more troublesome. Cottonseed meats, for example, are extracted twice, being removed from the extraction thimble and ground between times. Further
more, almost all of these complete extraction methods are slow, the average elapsed time necessary being about 5 to 6 hours. But their greatest disadvantage lies in the fact that a laboratory and either a chemist or a specially trained technician are required for their operation. The writer believes that analyses of oil- and fat-bearing substances would be made much more frequently if methods were avail
able which, while accurate, would be much less difficult in operation, and above all if sufficiently rapid so that the result could be made known to the plant operator in time for him to make use of the information.
Methods which are rapid, accurate, and sufficiently simple so that non-technical persons can operate them have been devised. The time required varies from 10 minutes with cottonseed meal to 35 minutes with finely ground chocolate liquor or ground beef scrap (“ cracklings” ). Their accuracy is at least as great as that claimed for the official methods and their simplicity is such that a laboratory boy can become adept after a few hours’ practice.
Im portance of T im e Factor
Fats and oils of vegetable origin do not occur naturally in a pure state and analysis is necessary to assay the value of the raw material. As it is usually possible to wait at least several hours without inconvenience for analyses of products not yet in process, the time required for such a test is ordinarily not of paramount importance. But during the processes for isolation of the desired product— as, for example, by pressure methods— prompt knowledge of the fat content of the mate
rial, including the expressed residues, is necessary if efficient operation is to be maintained. The efficiency of a press must be known quickly if avoidable losses are to be stopped. In certain cases, where it is desired to press to a definite fat content of non-fatty residue, as in the case of breakfast cocoa
1 R eceived Septem ber 8, 1930. to the consumer, as the digestibility of cocoa decreases slightly with increase of fat content. Y et the author has known of object in mind. Most of them are variations of the standard method mentioned above. Some are very slow, a few are quite rapid, but all are subject to the objection that a skilled chemist is required to obtain significant results. With few exceptions the idea has always been to isolate the actual fat present and to weigh it.
The most exact results are said to be given by Heller’s (S) method, which consists of a 24-hour extraction in a Soxhlet apparatus. Kreutz’s (10) variation involves a digestion with chloral hydrate previous to extraction with ether. Kooper (9) modified the well-known method of Gerber for milk, a centrifugal method similar to the Babcock, and by using sodium salicylate solution to dissolve the protein and butanol as a clarifying agent he applied this rapid method to cocoa.
Butyrometers somewhat similar to the Babcock apparatus are used. This method works well with dairy products such ammoniacal and alcoholic suspension before the ether extrac
tion, a mixture of petroleum and ethyl ethers being employed.
Gephardt3 speeded up these extraction methods by finding that good results are obtained by the use of only 1 gram of sample and by a very short extraction and washing of the material on a Gooch crucible. The method is rapid and probably accurate, but involves considerable skill.
A method devised by Hughes (7) is popular in some circles because of its rapidity. It consists of successive extractions of the fat-bearing substance with ether, centrifuging the ether layer after each extraction. Low results are obtained if the extraction has not been complete; on the other hand, high results are likely if the centrifuging of the finely divided par
ticles is less than perfect. A variation of the extraction method consists of the S. B. P. procedure, using trichloro- ethylene. A larger sample (8 to 10 grams) is taken and an aliquot of the extract is evaporated and weighed in the usual manner. The method is fairly rapid, but the technic is involved.
* This method is in use b y certain manufacturers, but so far as the writer is aware it is not described in the literature.
October 15, 1930 IN DU STRIAL AN D ENGINEERING CHEMISTRY 411 than the earlier official procedures. Heiduschka and Muth (4) have devised glass apparatus for extraction and evapora
tion methods which shorten the number of manipulations and transfers of solution. The Gooch crucible is designed to serve also as a weighing flask for the sample, and the dis
tilling flask is also fitted with a cover and is sufficiently small to act as a weighing flask for the extracted fat.
Richter {1/i) and Herty (<?) broke with tradition, so far as total fat estimation is concerned, and devised methods which do not depend upon weighing the fat actually present in the sample. They recognized that solution of a second substance in a liquid alters the physical properties of the liquid, usually in direct proportion to the amount of substance dissolved.
Therefore, if the change in that property can be sufficiently accurately measured,' the amount of added substance can be determined. Richter’s method depends upon the change in refractive index, Herty’s upon the change of specific gravity.
Richter used as dissolving liquid a mixture of ether, alcohol, and trisodium phosphate. The substance the fat content of which was to be determined was extracted with this mix
ture, the clear solution, obtained after warming, adjusted exactly to a definite temperature (17.5° C.), and examined under a Zeiss refractometer. From the refractive indices of solvent, solution, and the pure fat, the amount of fat present in solution, and consequently the amount present in the original sample, may be calculated. One difficulty with this method lay in the very great volatility of the ether and alcohol of the solvent. Evaporation of these substances during the operation will render this method inaccurate. To overcome this objection, Wesson {16) proposed the use of Halowax, a trade name for chlorinated naphthalenes. As certain of these products (impure monochloro compounds) are liquids, they can be used instead of the original mixture of Richter.
Halowax is non-volatile and otherwise suitable for this method, but the commercial product varies in composition and properties. The refractometer method has been further elaborated by Coleman and Fellows {1) to include a number of fat-bearing materials. Before using this method the Halowax must be standardized— i. e., a table showing the variation of refractive index with increasing percentages of the oil or fat must be developed before the particular batch of solvent may be used. According to Coleman and Fellows,
“ it is necessary to prepare for each new lot of the solvent a standard conversion table for each kind of oil-bearing mate
rial on which the test is to be made.” Furthermore, for certain materials, such as chocolate liquor, an involved process of preparation is necessary. This substance must be grated, melted, chilled, and grated again before the analysis can be run. The temperature of the solution must be carefully adjusted before the refractometer reading is taken. How
ever, when all these precautions are taken, a skilled operator can obtain results in very short intervals.
Herty’s {6) method consists of solution of the fat by means of carbon tetrachloride and analysis of the solution by the use of a Westphal balance. Owing to the volatility of this solvent, its evaporation during the operation will give high results. Here again the specific gravity of the solvent will vary over an appreciable range, and in contact with various materials will hydrolyze and form various degradation products which very much alter the gravity. Consequently, the solvent requires frequent checking and standardization, the operation requires considerable skill to avoid a change in specific gravity during the interval, and the Westphal balance is not without objection.
Experimental M ethod
Quantitative solution of a substance is considerably more rapid in many circumstances than quantitative isolation.
The latter involves the wTashing of a solution from solids mixed therewith. Theoretically this is an endless process approaching infinity as a limit. Practically a definite number of washings will prove sufficient for most purposes, but one source of error is insufficient washing. Another and more important one is the removal of the solvent. This is supposed to be done until constant weight is reached, but materials surface and not occluded in tissue cells, quantitative solution with a good solvent is extremely rapid.
For example, without the application of heat all of the cocoa butter present in a sample of cocoa wall enter solution in a chlorinated hydrocarbon in 3 minutes or less. It should be remembered in this connection that solution by extraction with petroleum ether in a Soxhlet appara
tus is also effected cold, the ether drip effected simultaneously from every par
ticle of the substance.
The fact that solution of a substance alters the physical properties of the sol
vent is well knowm. Herty’s {6) method is based upon this principle. An aliquot of the solution is sufficient to examine the change in physical property effected, and this change is a measure of the amount of solute present in the original sample. It is necessary to measure the degree of this
change with sufficient accuracy so that the Figure i —Hydrom- substance, in this case oil or fat, may be C h o c o la te
estimated as closely as the best of the
methods now existing. Specific gravity was selected as the physical property, because it can be measured by hydrome
ters which are simple to use and which can be manu
factured to be accurate to 0.0001.
o-Dichlorobenzene was selected as the solvent. It has the advantages of being non-inflammable, very slightly volatile at ordinary temperatures, and not too expensive. Further, the difference in gravity between it and a fat is so great that small additions of fat cause a marked change in the gravity of the solution, thus permitting accurate results. Solutions of fat in this solvent may be permitted to stand exposed to the atmosphere for 24 hours with no observable change in specific gravity. As commercial o-dichlorobenzene is not uniform in properties, it is especially adjusted by the manu
facturers of the analytical equipment so that uniform results may be obtained.
The analytical balance was discarded, as men accustomed
412 A N A L Y T IC A L EDITION Vol. 2, No. 4
Vacuum
to handling tons of product in the plant or laboratory boys unfamiliar with careful technic could not be expected to use so delicate an instrument. Accordingly a torsion balance accurate to 0.1 gram was employed, using a sample of 100 grams in order to maintain the accuracy of weighing to 0.1 per cent.
The methods as developed consist of the solution of the fat with a definite weight of solvent, filtration of an aliquot from the insoluble residue, and determination of the change of specific gravity by means of special hydrometers accurate to 0.0001 specific gravity, and reading directly the percentage of fat present. Upon this principle are based methods for the determina
tion of total fat in chocolate products, such as liquor, sweet and milk coatings, cocoa powder, shells, expeller cake, and nibs. Methods have also been devel
oped for the determination of the oil in cottonseed meal and the tallow in ground meat scrap. Other develop
ments are in process.
Procedure for Chocolate Products A special aluminum beaker and stirring rod is placed on the right-hand pan of a torsion balance. A brass weight equal to the weight of the beaker and rod plus 100 grams is placed on the left pan and the sample intro
duced into the beaker until the scale is in equilibrium. A dash pot is used on sy the newest balances which damps the (s/ ---) vibration and brings the pointer to
rest very quickly. After 100 grams of sample, either cocoa powder or molten chocolate liquor or coating, have been thus weighed out, about 2 inches (5 cm.) of the occasional stirring. This procedure dissolves all of the cocoa butter present. A Büchner funnel is now prepared with a but should not exceed 8 to 10 minutes. The funnel is removed from the filtering cylinder, and one of the special hydrometers (Figure 2) is inserted in the solution of cocoa butter in the solvent. The percentage of cocoa butter present in the sample is read directly from this hydrometer. These hy
drometers are standardized at 20° C., but the temperature- correction scale makes accurate readings possible at any point between 17° and 23° C. Only a very approximate temperature adjustment is therefore necessary. If the tem
perature is too low, the mercury will not appear on the lower scale, and the solution in the cylinder can easily be warmed
The technic used for cacao nibs, shell, and expeller cake is
F ig u re 2— F ilte r in g th e
cedure is omitted. The operation subsequent to the grinding requires 10 minutes. The method for ground meat scrap (cracklings) is similar, but in this case it is necessary to allow the mixture of sample and solvent to stand for 15 minutes, decanting onto the funnel, in order to secure a sufficiently rapid filtration.
Results
A comparison of the results obtained by these and other methods is given in the accompanying table.
T o t a l F a t D e t e r m in a t io n s b y N ew a n d O ld M e t h o d s b y analysis o f the product and remains constant for that particular grade of coating.
& T he expeller cake must be ground to 60 mesh before being analyzed.
Acknow ledgm ent
The painstaking and capable assistance of Manuel Honvitz in working out many of the details is gratefully acknowledged.
Literature Cited
October 15, 1930 IN D U S T R IA L A N D ENGINEERING CHEM ISTRY 413 alkali to form sodium iodate
31 + 6NaOH — >- NalO, +
and alkali, resulting in general in overoxidation and therefore high values. Modification of the procedure is recommended, based on the observation that alkali added slowly to a solution of an aldose sugar containing a small amount of iodine will tend, not to form sodium iodate, but to react with the sugar.
By addition of the iodine and alkali successively in small por
tions the concentration of sugar relative to the iodine-alkali or sodium hypoiodite is kept at a level favoring the sugar-oxida
tion reaction. The rapid formation of iodate, therefore, indi
cates the complete oxidation of the aldose to the monobasic acid. By this procedure only 2 ml. excess of 0.1 N iodine are required. (Compare the first, third, and sixth columns under Xylose in Table I.)
Previous methods have specified empirical time periods of various extent up to 30 minutes for the completion of the oxidation. However, using the proposed modification, the reaction may be taken as complete 2 minutes after the last of the reagent is added.
Iodate formation takes place more rapidly than the very slow oxidation of dilute solutions of ketoses and non-reducing sugars. (Compare the third and sixth columns under Lévu
lose and Sucrose in Table I.) These non-aldehydic sugars, therefore, do not interfere with the determination of aldoses.
The slow oxidation of the non-aldehydic sugars consumes hydrogen-ion concentration, oxidation of the aldonic acid and of ketones and non-reducing sugars, and the tim e of oxidation. By adding the standard iodine first and then the alkali, each in sm all fractions of the total volum es required, the reaction is com pleted rapidly and a precision of 0.2 to 0.3 per cent is obtained. Iodate form ation is an index of the com pletion of the oxidation of the aldoses.
sugar calculated on the basis of the alkali c o n s u m p tio n to sodium sulfate b y iodine in alkaline solution, viz.:
NajSjO, + 41, + lO N aO H ► 2Na,SO, + 8NaI + 5H ,0 With xylose an apparent overoxidation of about 10 per cent is obtained with 20 ml. excess of iodine and 30 ml. excess of alkali, whereas if the solution is acidified before titrating with thiosulfate the true overoxidation is found to be about 2 percent.
Levulose and sucrose give about 7.5 and 6 per cent apparent oxidation, respectively, and 3.5 and 2 per cent true oxidation calculated on the basis of four equivalents of iodine required for the oxidation of one equivalent of sugar as found experi
mentally. If the percentage oxidation is calculated on the basis of two equivalents of iodine required for oxidation, as found experimentally for aldoses, the above values are doubled.3 The free iodine and iodine combined as hypo
iodite may be determined with about 1 per cent error by satu
rating the alkaline solution with carbon dioxide and titrating with thiosulfate; the iodine combined as iodate is then ob
tained by acidifying with hydrochloric acid and completing the titration with thiosulfate.
The use of both indicators and the glass electrode has shown that at about pH 6.4 the reaction is very slow and requires 18 hours for only 34 per cent completion. When the pH is kept at about 9 to 10, the end point is reached within about 2 minutes after the last portions of the reagents are added.
The regulation of the pH is therefore of prime importance in both the analytical method and in the practical application of these reactions in the production of sugar acids now under way.
* T h e preliminary oxidation data reported by Slater and Acree ( / ) are too high ow ing to these tw o factors—nam ely, the titration of free iodine in alkaline solution, and the calculation o f the per cent oxidation of sucrose and levulose on the basis of tw o equivalents of iodine instead of four required per mol o f sugar.