ANALYSIS. oF MILK anp MILK PRODUCTS | LEFFMANN FOURTH EDITION Copyright N° COPYRIGHT DEPOSIT; i - : a bia Wy ' Sars Ws} i‘ Hy ‘ ‘ it Cah | nt ANALYSIS OF MILK AND MILK PRODUCTS LEFFMANN SANITARY RELATIONS OF THE COAL-TAR COLORS BY THEODORE WEYL AUTHORIZED TRANSLATION BY HENRY LEFFMANN I2mo. 154 Pages. Cloth, net $1.25 EXAMINATION OF WATER FOR SANITARY AND TECHNICAL PURPOSES Seventh Edition, Revised Illustrated. I2mo. Cloth, net $1.25 ORGANIC CHEMISTRY A COLLEGE TEXT-BOOK BY HeNryY LEFFMANN and CHartes H. LAWALL 12mo. Illustrated. Cloth, net $1.00 SELECT METHODS IN FOOD ANALYSIS BY HENRY LEFFMANN and WILLIAM BEAM Second Edition, Revised I2mo. Cloth, net $2.50 t Plate and 54 Illustrations ALLEN’S COMMERCIAL ORGANIC ANALYSIS VOLUME 8 CONTAINS: Methods of Analysis of Milk and Milk-Prod- ucts, Meat and Meat-Products, Proteins, Pro- teoids, Fibroids and Enzyms. 8vo. 696 Pages. $5.00 net ANALYSIS OF MILK AND MILK PRODUCTS BY HENRY LEFFMANN, M. D. PROFESSOR OF CHEMISTRY IN THE WOMAN’S MEDICAL COLLEGE OF PENNSYLVANIA AND IN THE WAGNER FREE INSTITUTE OF SCIENCE OF PHILADELPHIA; PATHOLOGICAL CHEMIST TO JEFFERSON MEDICAL COLLEGE HOSPITAL FOURTH EDITION, REVISED AND ENLARGED WITH ILLUSTRATIONS THE FIRST TWO EDITIONS OF THIS WORK WERE PREPARED AND ISSUED UNDER THE JOINT AUTHOR- SHIP OF HENRY LEFFMANN AND WILLIAM BEAM PHILADELPHIA r, BLARISTON 5 SON & CO, 1012 WALNUT STREET CopPyYRIGHT, 1915, BY HENRY LEFFMANN THE MAPLE PRESS YORK PA ©c1a491417 JUN 1619 hse PREFACE This book is intended as a guide to the analysis of milk and milk products in the routine of the commercial and _ food-inspection laboratory. Only processes of practical value have been given, and nothing has been said as to the food value of milk and its products, nor concerning the ef- fects of the several adulterants that may be detected. A notable portion of the text has been taken from Volume 8 of the Fourth Edition of Allen’s Commercial Organic Analysis. I am indebted to the courtesy of Messrs. P. Blakiston’s Son & Co. for permission to use this matter. An interesting point is to be noted in the com- parison of this edition with the first, issued about a score of years ago in association with Dr. William Beam. In that, a considerable part of the material was derived from the publications of foreign workers; in the present, American in- vestigations form the basis of many of the im- portant processes. “Westward the star of empire takes its way.” eke PHILADELPHIA. CONTENTS MILE. PAGE Analytic Dataianad Processes! 's jit e a seis hele 1-64 MiILkK PRODUCTs. Dp coer URN (SPS eT OFT MM ee aah ema ROE RRO PUM MERT MIRE reo CODMENSER IIIs) uke se SAPs Aine he) Si Ga se POLO} = pel ales (Pome Maat eile SUR HELLS eat AC MUN TAINAN 6 2 EN AROS Matin reh A NRie Ai: faa 97-109 Bermented, Male PrOGuUucts: oj). sos vies e) sete, be I10-I13 INDEX Vil iy en , h, tin ra ee MILK ANALYTIC DATA Milk, the nutritive secretion of nursing mam- mals, contains water, fat, proteins, sugar, and mineral matter. Cow’s milk is meant in all cases, unless otherwise stated. Milk as taken from the animal is generally termed ‘‘whole nile.” Fat.—This occurs in globules varying from 0.0015 mm. to 0.005 mm. in diameter, in a condition which prevents spontaneous coales- cence. It is peculiar among animal fats in containing a notable proportion of acid radicles with a small number of carbon atoms. Proteins —The nature of the proteins of milk has been much discussed, but it is now generally conceded that there are at least three forms, casein, albumin, and globulin, the casein being present in by far the greatest amount, and the globulin as traces only. CasEin.—Casein is probably in part in combination with phosphates. It is precipi- I 2 MILK tated by many substances, among which are acids, rennet, and magnesium sulfate, but not by heat. Acids precipitate it by breaking up the combination with phosphates. The action of rennet is complex and probably partly hy- hydrolytic, splitting the casein into several proteins, some of which are precipitated in the curd. Films of protein matter occur abun- dantly in milk, for which reason it is distinctly opaque, even when nearly all the fat has been removed by contrifugal action. The albumin of milk appears to be a distinct form, and is called lactalbumin. It is not precipitated by dilute acids, but is coagulated by heating to 70°—75°. The proportion in cow’s milk is usually from 0.35 to 0.50%, but col- ostrum may contain much larger proportions. Globulin is present only in minute amounts in normal milk, but colostrum may contain as much as 8%. It is coagulated on heating. Lactose.—This is a sugar peculiar to milk. Citric acid is a normal constituent of the milk of various animals. In human milk, the quantity is about o.5 gram to 1000 c.c.; in cow’s milk, from zr to 1.5 grams. It is not dependent on the citric acid present in the food. Enzyms.—Several enzyms occur in milk but they are chiefly known by effects and not as isolated substances. Some are proteolytic, others ANALYTIC DATA 3 are oxydases, that is, decompose hydrogen per- oxid and carry oxygen over to other substances. Lecithin is also a usual ingredient of milk. Nerking and Haensel found a range in cows’ milk from 0.03 to 0.11%. Mineral Matter—vThe ash of milk contains calcium, magnesium, iron, potassium, and sodium as chlorids, carbonates, sulfates, and phos- phates. It does not exactly represent the salts present in milk. Richmond has determined the ratio of the ash to the solids not fat in 135 samples of milk. This was found to range from 7.8 to 9.4%, but more usually from 7.8 to 8.5 (average 8.2) %. Many ashes were alkaline to turmeric, litmus, and phenolphthalein, the maximum alkalinity being 0.025% calculated as sodium carbonate. Human milk is notable for a low protein content hence the curd is less bulky and more friable than that from cows’ milk. The milk of all animals is subject to modification by breed, climate, season, feed, housing, exercise, time of lactation, and in human beings (and possibly in some other animals) by psychic influences. As regards the proportion of proteins and lactose, milks of the mare and ass agree closely with human milk. Normal milk is an opaque white or yellowish- white fluid, with an odor recalling that of the 4 MILK animal, and a faint sweet taste. The opacity is due largely but not entirely to the fat globules. The reaction of freshly drawn milk to litmus is usually alkaline, but is sometimes amphoteric; that is, it turns the red paper blue and the blue paper red. The sp. gr. varies between 1.027 and 1.035. It usually undergoes a gradual augmentation (sometimes termed Recknagel’s phenomenon) for a considerable time after the sample has been drawn. The increase may amount to two units (water being 1000). The sp. gr. becomes stationary in about five hours if the milk is maintained at a temperature below 15°, but at a higher temperature it may require twenty-four hours to acquire constancy. The change is not entirely dependent on the escape of gases. Unless collected with special care and under conditions of extreme cleanliness, milk always contains many bacteria, animal matter of an offensive character, such as epithelium, blood and pus cells, particles of feces, and soil. At ordinary temperature milk soon undergoes decomposition, by which the milk sugar is converted principally into lactic acid, and the proteins partly decomposed and partly coagu- lated. The liquid becomes sour and the fat is inclosed in the coagulated casein. In the initial stages of decomposition the proteins frequently ANALYTIC DATA 5 undergo transformations into substances which are the cause of the violent poisonous effects occasionally produced by ice-cream and other articles of food into the preparation of which milk enters. Boiling produces coagulation of the albumin, some caramelization of the sugar, and develops a greater facility of coalescence on the part of the fat globules. Enzyms are rendered inert and most microbes are killed. When milk is allowed to stand, some of the fat rises gradually and forms a rich layer, constituting cream. The proportion of cream depends on several conditions. The amount formed in a given time cannot be taken as a measure of the richness of the milk. Water added to milk Causes a more rapid separation of the cream. Centrifugal action separates nearly all of the fat. The following figures, given by D’Hout as aver- ages, show this effect: WHOLE SEPARATED CREAM ILK MILK Specific gravity..... 1032 1034 I0I5 otal solidee o> 14.10 9.6 26.98 AE ieee ese ys 4.70 5.05 3.32 SoeeE ace, ble edie 3.50 3.62 2.02 ME. Frc canoe ee 0.79 0.78 0.58 RM erro da atotes 2 5.05 0.20 21.95 Buttermilk is the residue after removal of the butter by churning. Vieth gives the following analyses: 6 MILK SOU iali Hanis) SURGE ede 9.03 0.63 8.40 0.70 8.02 0.65 v Monts 4 1.29 10.70 0.54 10.16 0.82 Whey or Milk-serum is the liquid freed from curd after precipitation by rennet or acids. In most cases it contains a notable amount of proteins, as shown in the following analyses by Cochran: MILK WHEY Total solids Solids not fat Total solids debins aed 9.13 6.62 2.51 9.27 9.13 6.1 2/03 14.05 8.35 6.62 2.33 Bos 7.61 5.98 1.63 8.91 8.71 6.50 2.21 The whey of any given milk has practically the same composition, whether taken from the original milk, skimmed milk, or cream. Average Proportion of Solids in Milk.—The most extensive data on this point are those obtained by Vieth. The total number of samples was 120,540. ‘The averages of the entire series are as follows: | age aS UIT Su aa RA et AEP Re AR 4.1% PN are irc SOUS, 2 Nei. oy iets nice Nate aE 8.8% Weta Sole ile sc Gita tetecairane Gide wee eee 12.9% Lythgoe gives a table of averages of composi- tion of 51 samples of genuine milk, each set of ANALYTIC DATA 7 averages being deduced by analysis of 10 samples. The following data are selected from this table. For explanation of the figures in the last column see page 42. Sottps REFRACTION gene) 1) EAS, yp ames Niaeaees fe) NET Oe ge 15.70 6.08 i ae A gO O57 70, PO. O08 38.1 15.00 62a 750 ALee |) On 7G.) QO. 30 38.3 14.50 Bese sun hes wOngz.. Qi 20 38.3 14.00 Ae FOR SSE bas OS) Oe 7a: Oeae 38.5 13.50 AGE eay.)) Aang 0.75). \ 8.80 35). 1 13.00 = ls Mae Se er AA He SMR 8 By be 6 5 37.9 12.50 BOO BiBa Asean OL7 3: i C25I 38.0 12.00 BARN i 2UBS 14 206). Os7A! | SLRS 37-7 II.50 BL Asi 2 OR ht OO i OngOw ea le ey eS 11.00 2 G2)) 200d) ANOS (Ong eg ge a7 .0 10.70 2190") 2060.) \\ecag's Ov7E |)” ‘7-80 36.4 From these figures Lythgoe derives the rule that differences in proportion of solids not fat in unadulterated milks are principally due to dif- ferences in the amount of proteins. Lactose and ash are fairly constant. On these facts depend recently introduced methods of detecting water- ing milk, as will be pointed out later. Colostrum.—This is the secretion in the early stages of lactation, and differs from ordinary milk. It contains characteristic structures, known as colostrum corpuscles, and usually contains much less fat than fully developed milk, but a larger proportion of proteins. Colo- strum coagulates on boiling. Lactose is in small amount. 2 ANALYTIC PROCESSES Specific Gravity—The sp. gr. of milk rises gradually for some time after it has been drawn, and the determination is to be made only after this action has ceased. This will require about five hours after the milk is drawn, if it has been kept 15°, but at a higher temperature it will be necessary to allow at least twelve hours. For all other determinations the milk must be ana- lyzed as soon as possible. The following figures, published by Bevan, show that a considerable loss in total solids may occur in twenty-four hours: TOTAL SOLIDS Loss Evaporated immediately ... 5L.73 Evaporated after 24 hours, 10.79 0.94 Evaporated after 48 hours, 10.38 1.35 Evaporated after 120 hours, 9.42 2.05 The decomposition is very irregular, and it is not possible to determine, by estimation of the lactic acid or other products, the original compo- sition of the milk. Air-bubbles are held rather tenaciously by milk, and care must be taken in mixing, preparatory to taking the sp. gr., to avoid as far as possible 8 ANALYTIC PROCESSES 9 the inclosure of the air, and to allow sufficient time for the escape of any bubbles that may be present. Sp. gr. is understood to be taken at 15.5°; samples should be brought near to this. If at a few degrees above or below, it will suffice to make the determination at once and obtain the correct figure by reference to the annexed table. The sp. gr. of normal milk ranges between 1.027 and 1.035. The figure alone does not indicate the character of the sample, but taken in conjunction with the figure for fat or for total solids, it is of value as a check on the results furnished by other determinations. The simplest method of determining sp. gr. is by the lactometer, a delicate and accurately gradu- ated hydrometer. The instrument must be 1m- mersed carefully so as not to wet the stem above the point at which it will rest. Its accuracy should be verified by immersion in distilled water at 15.5° and milks of known sp. gr. More accurate determinations may be made with a balance. A special form, the Westphal balance, is adapted to the determination of sp. gr. only, the weights being so arranged that a simple enumeration of them gives the gravity directly. The cheap forms of this in- strument are not satisfactory, but some made by German houses are excellent. The ordinary Io MILK Find the temperature of the milk in one of the horizontal lines and the sp. gr. in the first vertical column. with this and the temperature the correct figure is given. ae Sp.Gr. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 pi ds 50 20.2 21.2 223 232 24.1 25.1 26.1 27.0 51 20.3|20.3 21.3/21.3 22.3|22.3 23.3/23.3 24.2/24.3 25.2/25.2 26.2/26.2 27.1/27.2 28.0 29.0|29.1|29.1|29.2 29.9 30.9 31.8 32.7 33.6 28.1|28.2 30.0/30.1 31.0131.1 31.9|32.0 32.9|33.0 33.8/33-9 10.0 I0.5|1I.1 52 | 53 54 20.4|20.5 21.4/21.5 22.4/22.5 23.4/23.5 24.4|24.5 25.3/25.4 26.3|26.4 27.3)27-4 28.3/28.4 29.3 30.2 at.2 32.1 33,1 34.0 11.6 39.3 31.3 32-3 33.2 34.2 12.2 55 20.6 21.6 22.6 23.6 24.6 25.5 26.5 27.5 28.5 29.4 30.4 31.4 32.4 33-3 34.3 n2.7 56 |57 |58 20.7|20.8/20.9 21.7|21.8|21.9 22.7|22.8|22.8 23.6/23.7|23.8 24.6|24.7/24.8 25.6/25.7/25.8 26.6/26.7|26.8 27.6|27.7/27.8 28.6/28.7|28.8 29.6|29.7/29.8 30.5|30.6/30.8 31.5/131.6|31.7 32.5|32.6|32.7 33-5|33-6/33.7 34-5|34-6134.7 In the same line 20.9 21.9 22.9 23.9 24.9 25-9 26.9 27.9 28.9 29.9 39.9 31.9 32.9 33-9 34-9 59 |60 | 61 62 21.0|\21.1/21.2 22.0|22.1/22.2 23.0|23.1/23.2 24.0/24.1|24.2 25.0|25.1/25.2 26.0/26.1/26.2 27.0|27.1/27.3 28.0/28.1/28.3 29.0|29.1/29.3 30.0/30.1/30.3 31.0/31.2|31.2 32.0/32.2132.3 33-0133-2/33-3 34.0/34.2/34.3 35-0/35-2/35-3 13.3|13.8/14.4/15.0)15.5|16.1|16.6 analytic balance may also be used. A plummet consisting of a thick glass rod (or short sealed tube, weighted with mercury) of a bulk of about To c.c. is suspended from the hook of the balance by means of fine platinum wire and the weight ascertained. It is then submerged in distilled water and the weight also noted. The water is contained in a narrow upright cylinder resting ANALYTIC PROCESSES Tz Find the temperature of the milk in one of the horizontal lines and the sp. gr. in the first vertical column. In the same line with this and the temperature the correct figure is given. 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 71 | 72 | 73 | 74 | 75 -3/21.4/21.5/21.621.7/21.8/22.0/22.1/22. 2/22. 3\22. 4/22. 5\22.6 -3\22. 4/22. 5\22.6/22.7/22.8/23.0 1/23. 2/23.3|23.4/23.5/23.7 23. 3\23.4/23.5|23.623.7|23.8/24.024.1\24.2/24.3/24.4/24.6|24.7 -3|24.4/24.5/24.624.7/24.9/25.025.1/25.2125.3125.525.6/25.7 -3\25.4/25.5|25.6125.725.9/26.0 1'26.2/26. 4/26. 5/26.6|26.8 26.3/26.5/26.6/26. ,O127. 0127... 1127: 27.4/27.5/27.6|27. .8/28.0/28.1/28. 7 8 Ae 27 a ra 727.8 28.4/28.5/28.6\28. 728.829.0729. 1\29. 829.9 830.9 3)*7-4/27-5/27-7 28 .3/28.4/28.6/28.7/28. 29.4/29.5/29.7/29.8 4130. 5/30. 7/30.9 5131.6/31.8 29.4/29.5/29.6/29. 30. 4/30. 530.730. 30. 4/30. . 7/30. 31.5131. .8131.9/32. Gite LN . 4/32. 5/32. 6132. 8132.0 -4/33 .6133.7|33 .923.0 -5/34.6/34.7/34.9/35.1/35. .5135-6/35.8136.0/36.1 . 5136. 7136. 8137.0137.2 LS LT | | ee | me | ee | es ce toe | Geetha fees tenes | Seen ee | pete fe 17.2|17.7/18.3|18.819.4|20.020. 5\21. 1/21 .6\22. 2/22. 7/23. 3/23.8 (eS) xs to wW ars Ww O rs nonin p&p A on a bench or support above the scale pan. The loss of weight of the plummet is, of course, the weight of the bulk of water that it displaces. The sp. gr. of any sample can be determined by weighing the plummet immersed in the sample and dividing the loss in weight by the loss in water. The quotient is the sp. gr. The ordinary pyknometer is not convenient I2 MILK on account of the liability of the upper layer of the liquid to be richer in fat than the lower; the overflow, therefore, does not represent the mixture. Total Solids.—This determination may often be made with sufficient accuracy for practical purposes by evaporating a measured volume (e. g.. 3 or 5 c.c.) in a shallow nickel dish from 5 to 8 cm. in diameter. Nickel crucible-covers are suitable. The thin glass (Petri) dishes used for microbe culture are convenient. When greater accuracy is required, and especially when the ash is to be determined, platinum dishes are the most satisfactory, but owing to the present price of this metal, quartz dishes are now much used. Either the translucent or transparent quartz is suitable, the former being less expensive. Good results may be secured as follows: A flat dish, 3.5 cm. in diameter, with sides 0.5 cm. high, is provided with a thin flat watch-glass cover that fits rather closely. The total weight of the cover and dish is noted. 2 or 3 c.c. of the sample are run into the dish from the pipet, the watch- glass placed on, and the weight taken as rapidly as possible. The glass prevents appreciable loss from evaporation during an ordinary weigh- ing. The cover is removed, the dish heated on the water-bath or in the water-oven, and weighed from time to time (with cover on it) until the ANALYTIC PROCESSES 13 weight is sensibly constant. The percentage of residue can be easily calculated. About three hours may be required to secure constant weight. When high accuracy is not essential, it will suffice to measure the milk. Vieth advised a pipet graduated to deliver 5 grams, and found that, working with whole and skimmed milk, under the ordinary variations of temperature, the error will not exceed o.1 on the total solids and less on the fat. The pipet should have a rather wide opening so that no cream will be retained. The Massachusetts State Board of Health has for many years used the routine method of evaporating 5 grams for two hours ina flat plati- num basin over boiling water. Phe A..OvA. C: method is: Heat at 100° to constant weight, about 3 grams in a tared plati- num, aluminum or tin dish of 5 cm. diameter, with or without the addition of 15 to 30 grams of sand. Cool and weigh. Ash.—tThe residue from the determination of total solids is heated cautiously over the Bunsen burner, until a white ash is left. The result obtained in this manner is apt to be slightly low from loss of sodium chlorid. This may be avoided by heating the residue sufficiently to char it, extracting the soluble matter with a few c.c. of water, and filtering (using paper extracted 14 MILK with hydrofluoric acid). The filter is added to the residue, the whole ashed, the filtrate then added, and the liquid evaporated carefully to dryness. The ash of normal milk is about 0.7% and faintly alkaline. A marked degree of alka- linity and effervescence with hydrochloric acid will suggest the addition of a carbonate. The method of the A. O. A.C. is as follows: In a weighed dish put 20 c.c. of milk from a weighing bottle; add 6 c.c. of nitric acid, evaporate to dryness, and burn at a low red heat till the ash is free from carbon. Fat.—Many methods for fat determination have been devised. The following will suffice for all practical work: Adams’ Method.—This consists essentially in spreading the milk over absorbent paper, drying, and extracting the fat in an extraction apparatus; the milk is distributed in an extremely thin layer, and by a selective action of the paper the larger portion of the fat is left on the surface. A paper, manufactured especially for this purpose by Schleicher & Schuell, is obtainable in strips of suitable size. Each of these yields to ether only from o.oo1 to 0.002 gram of extract. Coils made of thick filter-paper, cut into strips 6 by 62 cm., are thoroughly extracted with ether and alcohol, or the weight of the ex- tract corrected by a constant obtained for the ANALYTIC PROCESSES I5 paper. From a weighing bottle about 5 grams of the milk are transferred to the coil by means of a pipet, care being taken to keep dry the end of the coil held in the fingers. The coil is placed, dry end down, on a piece of glass and dried for one hour, preferably in an atmosphere of hydrogen; it is then transferred to an extraction apparatus and extracted with absolute ether, petroleum spirit of boiling-point about 45° or, better, carbon tetrachlorid. The extracted fat is dried and weighed. The above procedure is very satisfactory, but the drying in hydrogen may usually be omitted. After the coil has received at least twenty wash- ings, the flask is detached, the ether removed by distillation, and the fat dried by heating in an air-oven at about 105°, and occasionally blowing air through the flask. After cooling, the flask is wiped with a piece of silk, allowed to stand ten minutes, and weighed. Richmond states that to perform a rigidly accurate determination attention to the following points isnecessary: The ether must beanhydrous (drying over calcium chlorid and distilling is sufficient). Schleicher & Schuell’s fat-free papers should be used, and one should be extracted without any milk on it, as a tare for the others. Four or five hours’ extraction is necessary, and 16 MILK the coils should be well dried before extraction is begun. Thimble-shaped cases made of fat-free paper are now obtainable and are convenient for holding the absorbent material on which the milk is spread. The fine texture prevents undissolved matter escaping. A case may be used repeatedly. Sour milk may be thinned with ammonium hy- droxid before taking the portion for analysis. Babcock Asbestos Method.—This is recom- mended by the A. O. A. C.: Provide a hollow cylinder of perforated sheet metal 60 mm. long and 20 mm. in diameter, closed 5 mm. from one end by a disk of the same material. The per- forations should be about o.7 mm. in diameter ando.7 mm.apart. Fill the cylinder loosely with from 1.5 to 2.5 grams of freshly ignited woolly asbestos free from fine or brittle material. Cool in a desiccator and weigh. Introduce a weighed quantity of milk (about 4 grams) and dry at 100°. The cylinder is placed in the ex- traction tube and extracted with ether in the usual way. The ether is evaporated and the fat weighed. The extracted cylinder may be dried at 100° and the fat checked by the loss in weight. A higher degree of accuracy is secured by per- forming the drying operation in hydrogen. For thorough extraction, especially with diffi- culty soluble materials and volatile solvents, the ANALYTIC PROCESSES 17 continuous extraction apparatus devised by Szombathy, but commonly called the Soxhlet tube, is most suitable. The material may be placed in a fat-free paper thimble and covered with a plug of cotton to prevent loss of fine particles. In place of the cotton plug, a porcelain or platinum Gooch crucible may be used, as shown in the cut. The top of the thimble should be a short dis- tance below, and the top of the crucible a short distance above, the bend of the siphon. The thimble should be supported by a section of glass tubing, 1 to 2 cm. long, with rounded edges; the edge on which the thimble rests should be a little uneven to prevent a close joint, which would hinder the siphoning of some of the liquid. Alundum cylinders will probably be useful. Loss of solvent by leakage often occurs. It may be diminished somewhat by soaking the corks in rather strong hot gelatin solution, draining them quickly and then exposing them for some hours to formaldehyde vapor. The solvents most generally employed are ether and petroleum spirit, but carbon tetra- chlorid is well adapted for extraction purposes as it has high solvent power for fats and is not easily inflammable. } When extraction is completed, the carton and 18 MILK materials may be removed from the tube, and, replacing the parts of the apparatus, much of the solvent may be redistilled into the extractor, thus recovering the liquid. Care must be taken not to distil the contents of the flask closely or heat strongly, lest some of the more volatile of the dissolved matters pass into the distillate. Roese-Gottlieb Method.—This is now being used for milk-products as well as for milk. For de- tailed description, see page 72. Centrifugal Methods.—Although almost all the fat of milk may be separated by the centrifuge, the emulsion is not destroyed and the volume of cream is merely suggestive as to the fat-content of the milk. To obtain a clear fatty layer in condition for close measurement it is necessary to use chemicals. The methods at present most employed depend essentially on one devised by Gustaf DeLaval, who took out a patent in Sweden for the use of a mixture of twenty volumes of strong acetic acid and one volume of strong sulfuric acid. This mixture coagulates and then dissolves the proteins, destroys the emulsion, but does not otherwise affect the fat and does not act on the lactose. By brief whirling in a cen- trifuge the fat collects in a clear sharply defined layer. DeLaval took out patents in several countries subsequent to the above date. Leffmann and Beam devised a method in which ANALYTIC PROCESSES 1g a small amount of amyl alcohol with an equal volume of hydrochloric acid was added to the milk, and the proteins thus coagulated dissolved by strong sulfuric acid. About the same time Babcock devised a process in which sulfuric acid was used alone. Subsequently Gerber published a process in which the essential feature of the Leffmann-Beam method, namely, the use of amyl alcohol, was advised. The test-bottles have a capacity of about 30 c.c. and are provided with a graduated neck, each division of which represents 0.1% by weight of butter fat. 15 c.c. of the milk are measured into the bottle, 3 c.c. of a mixture of equal parts of amyl alcohol and strong hydrochloric acid added, mixed, the bottle filled nearly to the neck with concentrated sulfuric acid, and the liquids mixed by holding the bottle by the neck and giving it a gyratory motion. The neck is now filled to about the zero point with a mixture of sulfuric acid and water prepared at the time. It is then placed in the centrifugal machine, which is so arranged that when at rest the bottles are in a vertical position. If only one test is to be made, the equilibrium of the machine is maintained by means of a test-bottle, or bottles, filled with a mixture of equal parts of sulfuric acid and water. After rotation for from one to two minutes, the 20 MILK fat will collect in the neck of the bottle and the percentage may be read off. It is convenient to use a pair of dividers in making the reading. The legs of these are placed at the upper and lower limits respectively of the fat, allowance being made for the meniscus; one leg is then placed at the zero point and the reading made with the other. Experience by analysts in various parts of the world has shown that with properly graduated bottles the results are reliable. As a rule, they do not differ more than 0.1 % from those obtained by the Adams process, and are generally even closer. For great accuracy, the factor for correcting the reading on each of the bottles should be de- termined by comparison with the figures obtained by the Adams or other standard process. Cream is to be diluted to exactly ten times its volume, the sp. gr. taken, and the liquid treated as a milk. Since in the graduation of the test- bottles a sp. gr. of 1.030 is assumed, the reading must be increased in proportion. A more accurate result may be obtained by weighing in the test-bottle about 2 c.c. of the cream and diluting to about 15 c.c. The read- ing obtained is to be multiplied by 15.45 and divided by the weight in grams of cream taken. The mixture of fusel oil and hydrochloric acid seems to become less satisfactory when long ANALYTIC PROCESSES 2E kept. It should be clear and not very dark in color. It is best kept in a bottle provided with a pipet which can be filled to the mark by dipping. Rigid accuracy in the measurement is not needed. [The Leffmann-Beam method is often erro- neously called the ‘‘Beimling’’ method, but Beimling was merely the deviser of a cheap centrifuge. To protect the interest of a manu- facturer who had invested in the Beimling machine under the impression that it was a practicable method for fat estimation, it became necessary for Leffmann and Beam to take out a patent (now expired) and assign the same to this investor. | Calculation Methods.—Several investigators have proposed formule by which when any two of the data, sp. gr., fat, and total solids, are known, the third can be calculated. These differ according to the method of analysis employed. That of Hehner and Richmond, as corrected by Richmond, was deduced from results by the Adams method of fat extraction. It 1s: T =0.25G + 1.2F + 0.145 in which T is the total solids, G the last two figures of the sp. gr. (water being 1000), and F the fat. Patrick has proved that with American milks the constant should be dropped, the formula reading: T= 0.05'G +10 F 22 MILK Babcock’s formula has been much used in the United States. It is adapted to calculating the solids not fat. In this formula g is the entire figure for sp. gr. referred to water as 1. AF lal Snf = (pte 1) x25 (100 — f) Babcock has also given a much simpler form adapted for total solids. This differs but slightly from Richmond’s. Total Proteins.— 3 types of processes are employed for this estimation: Calculation from the total nitrogen; precipitation and _ direct weighing; calculation from the ‘‘aldehyde-figure.”’ Milk contains appreciable amounts of non- protein nitrogen, but the fact is usually disre- garded. According to Munk, this may range, in cow’s milk, from 0.022 to 0.034%, and from 0.014 to 0.026% in human milk. By these figures, the average protein nitrogen in cow’s milk would be 94%, and in human milk 91%, of the total nitrogen. Kjeldahl-Gunning Method.—(Calculation from total nitrogen). Reagents: Potassium sulfate-—A coarsely powdered form free from nitrates and chlorids should be selected. Sulfuric acid.—This should have a sp. gr. 1.84 and be free from nitrates and ammonium. ANALYTIC PROCESSES 23 Standard acid.—‘/, Sulfuric or hydrochloric acid, the strength of which has been accurately determined. Standard alkali.—% /,, Ammonium hydroxid, so- dium hydroxid, or barium hydroxid, the strength of which in relation to the standard acid must be accurately determined. Sodium hydroxid solution.—5o00 grams should be added to 500 c.c. of water, the mixture al- lowed to stand until the undissolved matter settles, the clear liquor decanted and keptina stoppered bottle. It will be an advantage to determine approximately the quantity of this solution required to neutralize 20 c.c. of the strong sulfuric acid. Indicator.—Cochineal solution is recommended by the A. O. A. C., but methyl-orange and sodium alizarin-monosulfonate are satisfactory. Methyl- orange solution should be very dilute; 1 part in tooo. A drop is sufficient for 100 c.c. of liquid. Phenolphthalein is not well adapted to tritation of ammonium compounds. Digestion and distillation flasks.—Jena-glass round-bottomed flasks with a bulb 12.5 cm. long and 9 cm. in diameter, the neck cylindrical, 15 cm. long and 3 cm. in diameter, flared slightly at the mouth. Process 5 c.c. of the sample are placed in a digestion 3 24 MILK flask, 10 grams of powdered potassium sulfate and 15 to 25 c.c. (ordinarily about 20 c.c.) of the strong sulfuric acid are added and the diges- tion conducted as follows: ‘The flask is placed in an inclined position and heated below the boiling-point of the acid for from five to fifteen minutes, or until frothing has ceased. Excessive frothing may be prevented by the addition of a small piece of paraffin. The heat is raised until the acid boils briskly. A small, short- stemmed funnel may be placed in the mouth of the flask to restrict the circulation of air. No further attention is required until the liquid has become clear and colorless, or not deeper than a pale straw. When Kjeldahl operations are carried out in limited number, the arrangement used in my laboratory has been found very satisfactory. A double-Y, terra cotta drain-pipe, about 20 cm. internal diameter, is connected by an elbow directly with the chimney-stack. The digestion flasks are supported as shown in the rough sketch, figure 1 (not drawn exactly to scale). Two flasks can be operated at once. The central opening is convenient for other opera- tions producing fumes. Openings not in use are closed by circles of heavy asbestos. Apparatus for use when many determinations are made are figured in the catalogs of supply- ANALYTIC PROCESSES 25 houses. As corrosive vapors are given off, it must be placed under a hood; but a special form of apparatus is now made which does not require an escape-pipe. When the liquid has become colorless or very light straw yellow, it is allowed to cool, diluted with 100 c.c. of water if the smaller form of Fic." 2: flask has been used, the liquid transferred to the distilling flask, and the digestion flask rinsed with two portions of water, 50 c.c. each, which are also transferred to the distilling flask. With the larger form of flask the dilution is made at once by the cautious addition of 200 c.c. of water. Granulated zinc, pumice stone, or 0.5 26 MILK gram of zinc dustisadded. 5oc.c. of the strong sodium hydroxid solution, or sufficient to make the reaction strongly alkaline, should be slowly poured down the side of the flask so as not to mix at once with the acid solution. It is convenient to add to the acid liquid a few drops of phenol- phthalein or azolitmin solution, to indicate when the liquid is alkaline, but it must be noted that strong alkaline solutions destroy the former indicator. The flask is shaken so as to mix the alkaline and acid liquids and at once attached to the condensing apparatus. ‘The receiving flask should have been previously charged with a carefully measured volume of the ‘/, acid (10 c.c. diluted with distilled water to 100 cc. is a convenient amount). The distillation is con- ducted until about 150 c.c. have passed over. A small amount of indicator is added, the liquid, titrated with standard alkali, and the amount neutralized by the distilled ammonium hydroxid determined by subtraction. Each c.c. of */, acid neutralized is equivalent to 0.007 nitrogen. The nitrogen multiplied by 6.38 gives the total proteins. The distillation in this operation requires care, as the amount of ammonium hydroxid is determined by its neutralizing power, hence solution of the alkali of the glass will introduce error. Common glassis not satisfactory. Block ANALYTIC PROCESSES 27 tin is a good material. Moerrs found that Jena- glass tubes resist the action of the ammonium hydroxid. Distillates should be titrated promptly as alkali may be dissolved from the glass. A satisfactory condensing arrangement for general laboratory use is a copper tank of good size, through which several condensing tubes pass. Aldehyde Number.—The addition of formalde- hyde to milk increases the acidity by an action on the proteins. As commercial formaldehyde is always acid, the acidity must be either determined or neutralized in applying the following method. The application of the reaction to determination of proteins in milk is due to Steinegger. Rich- mond and Miller investigated the method and suggested the use of strontium hydroxid instead of sodium hydroxid. Richmond gives the following details: Toarrovrere. Of mike ag Teast Ete. OL a Ore a solution of phenolphthalein is added and the liquid neutralized with standard strontium hy- droxid solution. To the faintly pink liquid, 2 c.c. or more of 40% formaldehyde solution are added and the titration made to the same tint as the former. The strontium hydroxid required by the formaldehyde solution must be known, and this being deducted from that which was used in the titration and the remainder calculated 28 MILK to c.c. N/, acid per 1000 c.c. of milk will give the ‘‘aldehyd number.’’ Richmond finds that this multiplied by 0.17 gives in most cases a close approximation to the total proteins obtained by the Kjeldahl method. Calculation Method.—Olson has shown that in normal milks the proteins may be calculated with close approximation by the formula in which is protein and ¢ total solids. DETERMINATION OF SPECIAL PROTEINS.— Casein and albumin may be determined by Sébe- lein’s method: 20 c.c. of the sample are mixed with 4o c.c. of a saturated solution of magnesium sulfate and powdered magnesium sulfate stirred in until no more will dissolve. The precipitate of casein and fat, including the trace of globulin, is allowed to settle, filtered, and washed several times with a saturated solution of magnesium sulfate. The filtrate and washings are saved for the determination of albumin. The filter and contents are transferred to a flask and the nitrogen determined by the method described above. The nitrogen so found, multiplied by 6.38, gives the casein. The filtrate and washings from the determina- ANALYTIC PROCESSES 29 tion of casein are mixed, the albumin precipitated by Almén’s tannin reagent, filtered, and the nitrogen in the precipitate determined as above. The same factor is used. Almén’s reagent is prepared by dissolving 4 grams of tannin in 190 c.c. of 50% alcohol and adding 8 c.c. of acetic acid of 25%. In a mixture of milk and whey (prepared with rennet) in about equal parts, Richmond and Boseley found about 0.3% of albumoses not pre- cipitated by the copper sulfate nor by magnesium sulfate, but precipitable, along with the albumin, by a solution of tannin. The separation may be effected by diluting the filtrate from the magne- sium sulfate precipitation, acidifying slightly with acetic acid, and boiling, when the albumin will be coagulated and precipitated. The albumoses may be separated by filtering the solution and precipitating with tannin solution. The pre- cipitated proteins are best estimated by de- termining the nitrogen in the moist precipitate. The separation of the proteins may be effected, though less accurately, but the use of acetic acid, as recommended by Hoppe-Seyler and Ritt- hausen. Leffmann and Beam have modified the process to avoid the delay and trouble of washing the precipitate, as follows: 10 c.c. of the milk are mixed with saturated magnesium sulfate solu- 30 MILK tion and the powdered salt added to saturation. The mixture is washed into a graduated measure with a small amount of the saturated solution, made up to 100 c.c. with the same solution, mixed, and allowed to stand until the separation takes place. As much as possible of the clear portion is drawn off with a pipet and passed through a dry filter. An aliquot portion of the filtrate is taken, the albumin precipitated by a solution of tannin, and the nitrogen in the precipitate ascertained as above. The following are A. O. A. ©. methods: 1. Provisional Method for the Determination of Casein in Cows’ Milk.—The determination should be made when the milk is fresh. When it is not practicable to make the determination within twenty-four hours, add one part of formaldehyd to 2500 parts of milk and keepinacool place. 10 grams of the sample are diluted with about go c.c. of water at between 40° and 42°, 1.5 c.c. of a solution containing 10% of acetic acid by weight added, allowed to stand for five minutes, washed three times by decantation, pouring the washings through a filter, and the precipitate transferred completely to the filter. If the filtrate is not clear at first, it will generally become so in two or three filtrations, after which the washing can be completed. The nitrogen in the; washed precipitate and filter is determined by the ANALYTIC PROCESSES 31 Kjeldahl-Gunning method. The nitrogen, multi- plied by 6.38, gives the casein. In working with milk which has been kept with preservatives, the acetic acid should be added in small portions, a few drops at a time with stirring, and the addition continued until the liquid above the precipitate becomes clear or nearly so. 2. Provisional Method for the Determination of Albumin in Milk.—The filtrate obtained in the above operation is neutralized with sodium hydroxid, 0.3 c.c. of the 10% solution of acetic acid added, and the mixture heated for fifteen minutes. The precipitate is collected on a filter, washed, and the nitrogen determined. Van Slyke has pointed out that the casein can be approximately ascertained by multiplying the figure for total proteins by 0.8. Modified Proteins, Amino-derivatives and Am- monium Compounds.—The following procedures are given by Van Slyke. The filtrate from the albumin precipitate is heated to 70°, 1 c.c. of 5% sulfuric acid added, then solid zinc sulfate to saturation. The mixture is allowed to stand at 70° until the caseoses settle. The liquid is cooled, filtered, the precipitate washed with saturated solution of zinc sulfate slightly acidified with sulfuric acid and the nitrogen ascertained by the Kjeldahl method. 32 MILK For amino-derivatives and ammonium com- pounds, 50 c.c. of the milk are mixed in a flask marked at 250 c.c. with 1 gram of sodium chlorid. A 12% solution of tannin is added, drop by drop, until no further precipitation occurs. The mix- ture is diluted to the mark, shaken and filtered through a dry filter. For amino-derivatives, 50 c.c. of the filtrate are treated for nitrogen in the usual way. For ammonium compounds, too c.c. of the filtrate are mixed with magnesium oxid and about 50 c.c. distilled, the distillate being received in a known volume of standard acid. Large excess of magnesium oxid must be avoided. Lactose.—For this determination, A. O. A. C. employs Soxhlet’s method with the following reagents: Copper sulfate solution.—34.639 grams of pure crystallized copper sulfate are dissolved in water and made up to 500 C.c. Alkaline tartrate solution.—173 grams of pure sodium potassium tartrate and 50 grams of good sodium hydroxid are dissolved in water and the solution made up to 500 c.c. Sodium hydroxid “/,. 2s c.c. of the sample in a 500 c.c. flask are diluted with 400 c.c. of water and 1o c.c. of the copper sulfate solution and 8.8 c.c. ‘/, sodium hydroxid solution added. The mixture should ANALYTIC PROCESSES 33 still have an acid reaction and contain copper in solution. If this is not the case, the experiment must be repeated, using a little less of the alkali. The flask is filled to the mark with water, shaken, and the liquid passed through a dry filter. so c.c. of Fehling’s solution, obtained by mixing equal parts of the above copper sulfate and alkaline tartrate solutions, are heated to brisk boiling in a 300 c.c. beaker, 100 c.c. of the filtrate obtained as above added, and boiling continued for six minutes; the liquid then promptly filtered, and treated according to methods given below. The amount of lactose is calculated by the table on page 34 from the copper obtained by table. The figures for weights of copper between any two data given in the table may be calculated with sufficient accuracy for practical purposes by allowing 0.0008 gram of lactose for each o.oo1 gram of copper. The precipitated cuprous oxid is usually con- verted into free copper and weighed as such. Two methods may be employed for reduction: by hydrogen or by electrolysis. Reduction by Hydrogen.—The curpous oxid is collected on an asbestos filter. This isarranged most conveniently in a special filtering tube, which is shown in figure 2. The wider part is about 8 cm. and 1.5 cm. in diameter, the narrower portion about 5 cm. long and o.5 cm. in caliber. 34 MILK A perforated platinum disk is sealed in just above the point of narrowing. ‘The asbestos is placed on this disk, washed free from loose fibers, dried well, and the tube weighed. The filtering tube is attached to an exhaustion apparatus by passing narrower portion through the cork, and a COPPER LACTOSE CoPpPER LACTOSE || COPPER LACTOSE 0.100 0.072 0.205 0.151 0.305 0.228 0.105 0.075 0.210 0.154 0.310 0.232 0.110 0.079 0.215 0.158 0.315 0. 236 0.115 0.083 0.220 0.162 0.320 0.240 0.120 0.086 0.225 0.165 0.325 0.244 0.125 0.090 0.230 0.169 0.330 0.248 0.130 0.094 0.235 O.172 0.335 0.252 0.135 0.097 0.240 O.177 0.340 0.256 0.140 0.101 0.245 0.181 0.345 0.260 0.145 0.105 0.250 0.185 0.350 0.264 0.150 0.109 0.255 0.189 0.355 0.268 0.155 0.112 0.260 0.192 0.360 0.272 0.160 0.116 0.265 0.196 0.365 0.276 0.165 0.120 0.270 0.200 0.370 0.280 0.170 0.124 0.275 0.204 0.375 0.285 0.175 0.128 0.280 0.208 0.380 0.289 0.180 0.132 0.285 0.212 0.385 0.293 0.185 0.134 0.290 0.216 0.390 0.298 0.190 - 0.139 0.295 0.221 0.395 0.302 0.195 0.141 0.300 0.224 0.400 0.306 0.200 0.147 ANALYTIC PROCESSES 35 small funnel is fitted tightly in the top of the tube. The object of this funnel is to prevent the pre- cipitate collecting on the upper part of the tube. The lower end of the funnel should project several centimeters below the bottom of the cork through which it passes. The filtering apparatus must be arranged prior to the precipitation, so that the cuprous oxid may be filtered without delay. The pre- cipitate is transferred as rapidly as possible to the filter, well washed with hot water, alcohol, and ether successively, dried, and the cuprous oxid reduced by gentle heating in a current of hydrogen. When the reduction is complete, the heat is withdrawn, but the flow of hydrogen is continued until the tube is cold. It is then detached and weighed. Reduction of Copper by Electrolysis.—The fil- tration is performed in a Gooch crucible with an asbestos-felt film and the beaker in which the precipitation was made is well washed with hot water, the washings being passed through the filter, but it is not necessary to transfer all the precipitate. When the asbestos film is completely washed, it is transferred with the adhering oxid to the beaker; any oxid remaining in the crucible is washed into the beaker by use of 2 c.c. nitric acid (sp. gr. 1.42), added with a pipet. The crucible is rinsed with a spray of 36 MILK water, the rinsings being collected in the beaker. The liquid is heated until all the copper is in solution, filtered, the filter washed until the filtrate amounts to at least 100 c.c., and elec- trolyzed. Electrolytic apparatus has been constructed in a great variety of forms. When the opera- tion is carried out frequently, it is best to have an electrolytic table. A platinum basin holding not less than roo c.c. isused. A cylindrical form with flat bottom is convenient. It should rest on a bright copper plate, which is connected with the negative pole of the electrical supply. The positive pole should be also platinum, either a spiral wire, cylinder, or flat foil, Many operators use a funnel-shaped perforated ter- minal for the negative pole; in which case a glass beaker or casserole will be a suitable container, the positive terminal being placed within the negative. Four cells of a gravity battery will suffice for a single decomposition, and will operate two, but more slowly. It is usual to arrange the apparatus so that the operation may be continued during the night. When the elec- tricity is taken from the general supply of the laboratory, it is usually necessary to interpose resistance and to have some means of measuring the current-flow. This is sometimes done with ANALYTIC PROCESSES 37 a gas evolution cell and incandescent lamp, but an ammeter and adjustable rheostat are better. Lactose may be determined by the polarim- eter after removal of the fat and proteins, which is best effected, as recommended by Wiley, by acid mercuric nitrate solution. Wiley prepared this by dissolving mercury in twice its weight of nitric acid of 1.42 sp. gr. and adding to the solution five vol- umes of water, but Revis and Bol- ton advise that mercuric oxid ,. should be used. The A. O. A. C. optical method is as follows: For polarimeters reading to 100 for 26.048 grams sucrose (corre- sponding to 32.98 grams lactose), measure, in c.c., the amount ob- tained by dividing double this (2.e., 65.96) by the sp. gr., add roc.c. mercuric nitrate solution, make up to 102.6 c.c., shake, filter through a dry filter and examine in a 200 mm. tube. Half the observed reading will be the per- centage of lactose. For example, if the sp. gr. of the milk is 1.030, the amount taken will be 65.90 + 1.030 = 64 C.c. The allowance for volume of precipitate by making up to 102.6 c.c. is not accurate, except with closely skimmed milks. FIG, 2; 38 MILK The correction may be made more closely by calculating the actual volune of the precipitate by multiplying the fat-percentage by 1.075 (average specific volume of fat) and the protein- percentage by 0.8 (average specific volume of coagulated proteins), deducting the sum of these products from too c.c. and correcting the ob- served reading by proportion. For ordinary milk, the volume of the proteins from 65.96 grams may be taken at 1.68 c.c. Supposing the sample to contain 4.0% of fat and the polarimetric reading to be to, the calculation would be thus: 65.96 X 0.04 = 2.63 Amount of fat in milk taken 2.63 X 1.075 = 2.82 .c.c. Volume of fat in precipitate 1.68 c.c. Est. vol. of proteins in precipitate Soe 4.50 c.c. Total volume of precipitate 100 — 4.50 = 9.55 c.c. Actual volume of liquid. 100 :95.5::10:9.55 9.55 +2 = 4.75, per cent. lactose. The employment of a factor for correcting for the volume of precipitate may be avoided by Scheibler’s method of ‘‘double dilution,’’ in which two solutions of different volume are compared. The following is a summary of the method given by Wiley & Ewell: For polari- meters adapted to a normal weight of 26.048 sucrose, 65.82 grams of milk are placed in a 100 c.c. flask, 10 c.c. of the acid mercuric nitrate ANALYTIC PROCESSES 39 added, the flask filled to the mark, the contents well mixed, filtered, and a reading taken. A similar quantity of the milk is placed in a 200 c.c. flask and treated in the same way. The true reading is obtained by dividing the product of the two readings by their difference. If the observations are made in a 200 mm. tube the percentage is half the true reading. The instrument should be accurate, and great care taken in the work, or the results will be less satisfactory than by the method first described, in which an allowance is made for the volume of the precipitate. Mutltirotation.—When freshly dissolved in cold water, lactose shows a higher rotation than that given above. By standing, or immediately on boiling, the rotary power falls to the point mentioned. In preparing solutions from the solid, therefore, care must be taken to bring them to the boiling-point previous to making up to a definite volume. This precaution is unnecessary when operating on milk. Acidity.—Milk being often amphoteric to lit- mus, that indicator cannot be employed in estimating acidity. Phenolphthalein is usually employed. Several methods differing in details have been proposed. Probably the best is that of Thérner. In this, 10 c.c. of milk are diluted with 20 c.c. of water, a few drops of a dilute 4 40 MILK alcoholic solution of phenolphthalein added and the titration made with standard alkali. Thorner proposes that the number of c.c. required should be multiplied by 10 and the result termed the ““degree of acidity.’’ Fresh normal milk will show figures ranging from 16 to 18. When the degree of acidity is 23 or over, the sample will coagulate on heating. The process involves a slight error, in that the addition of a notable amount of water to a milk sample disturbs somewhat the relation of the phosphates and diminishes the acidity. It may be advisable to titrate the undiluted milk. If the number of c.c. used is multiplied by o.9 the lactic acid equivalent to the acidity of the sample is given in grams per 1000 C.c. DETECTION OF ADULTERATION By far the larger part of the laboratory work on milk is for assistance in the sanitary control of the supply, and the analyses are principally directed to the detection of the ordinary forms of adultera- tions. The most important of these are: skim- ming, watering and use of coloring, thickening and preserving agents. Skimming and watering are detected by determining fat and total solids; from these data the solids not fat are calculated. For the ordinary purposes of milk control, fat can be estimated with quite sufficient accuracy by centrifugal methods. The total solids may be estimated directly as described on page 12, or calculated from the sp. gr. and fat as indicated on page 21. Judgment whether a given sample has been skimmed or watered depends in many cases upon the standard for whole milk. Some irregularity of standards for fat and solids not fat exists, and the opinion of the analyst will be determined, therefore, by the standard of the locality. In most cases the standard for fat is between 3 and 4%, and that for total solids about 8.50%. As fat diminishes the sp. gr. of milk, and the AI 42 MILK other solids increase it, it is possible to take off a small amount of the former and add some water without disturbing the sp. gr., but, of course, the above analytical methods will detect this procedure. It is now admitted that, except in cases of wide departure from the usual limits, the adulteration of milk cannot be detected by the sp. gr. alone but the employment of a care- fully graduated lactometer is of use in routine milk inspection. Direct Detection of Added Water. Serum-refrac- tion.—Of late years several methods have been proposed for this purpose but most of them have no positive value and have not come into general use. The refractive index of the whey (milk- serum) offers a rapid and satifactory method for detecting watering. Several methods of pre- .paring this whey have been proposed, but Lythgoe has found, as the result of extended experience, the following to be satisfactory. Dissolve 7.25 grams of crystallized copper sulfate in water and dilute to 1000 c.c. If this solution does not refract 36 on the scale of the immersion refractometer at 20°, add water or copper sulfate until the desired result is obtained. To 8 c.c. of the copper solution add 32 c.c. of milk. Shake well and pour upon adry filter. When the filtrate begins to come through clear, change the receiver, pour the small quantity of cloudy filtrate upon DETECTION OF ADULTERATION 43 the filter and continue the filtration as usual. Refract the clear filtrate at 20°, by means of the Zeiss immersion refractometer. Areading below 36 indicates added water. The advantages of this method over the acetic acid method are as follows: It is quicker, heating of the samples is unnecessary, consequently there is no error due to evaporation. The range of differences in the refraction of pure milk is less. 10% of added water will reduce the refraction of high-grade milk below the minimum, but it takes 15% inthe acetic acid method. Lythgoe made analyses of 150 samples of milk of known purity by this method. The total solids ranged from 17.17 to 10.40%, the fat from 7.7 to 2.45%, the solids not fat from 10.50 to 7.5% and the refraction of the copper serum from 36.1 to 39.5. These refrac- tions were distributed as follows: REFRACTION NUMBER OF SAMPLES 39.0 to 39.5 6 38.0 to 38.9 66 37:0 baia7 9 65 36.1 to 36.9 13 150 See also table of refractions on page 7. As a result of extended experience, Lythgoe has recently given the following applications of some of the methods of milk analysis. The least variable constituents of milk are 44 MILK lactose and ash, both of which are valuable data in detecting added water. It is possible within reasonable limits to indicate by the total solids and fat whether a given sample has been watered or skimmed. No relation exists between the refraction of the (sweet) serum and the ash of the sour serum (see page 66), therefore, if both these data are below those of normal milk, added water is positively indicated. The ratio of protein to fat in normal milk is always less than 1. If the ratio exceeds 1, skimming is indicated. If the protein-fat ratio is less than o.7, or the percentage of fat to total solids is over 35, in samples having a low serum refraction, these may be declared watered, the refraction being not necessarily below the minimum for all samples of known purity. The sp. gr. of the sweet serum or its total solids may be used as a datum in place of the re- fraction; either will be a safe guide. Lowering of Freezing-point.—Several observers have shown that watered milk has a lower freezing- point than pure milk, and that the amount of depression has a definite relation to the amount of water added. One of the most recent state- ments on the subject is by J. W. Leather, who found the procedure very satisfactory for de- tecting watering in cows’ milk and that of the DETECTION OF ADULTERATION 45 India buffalo. He states that one observer has found that a depression to 0.537° indicates 2.3% of added water. The procedure requires special apparatus and careful manipulation; data from testing samples of known composition should be obtained before relying on it in important cases. Thickening Agents.—To conceal skimming and watering many thickening agents have been used. At least two instances of the use of brain matter have been reported. Dextrin, starch, sugar, salt, gelatin and agar have all been used. Brain matter can be easily detected by the microscope, starch jelly by the iodin test, dextrin by increased polarimetric reading, sodium chlorid by the increased chlorids in the ash. Agar is frequently used in certain milk products, especially the cheap ice-cream sold in American cities. Gelatin.—Stokes detects the presence of gelatin in cream or milkasfollows: toc.c. of the sample, 20 c.c. of cold water, and 10 c.c. of acid mercuric nitrate solution (page 37) are mixed, shaken vigorously, allowed to stand for five minutes, and filtered. If much gelatin is present, it may be difficult to get a clear filtrate. A portion of the filtrate is mixed with an equal bulk of saturated aqueous solution of picric acid. 46 MILK Gelatin produces a yellow precipitate. Picricacid will detect the presence of 1 part of gelatin in 10,000 parts of water. The picric acid solution should not give a precipitate with the nitrate solution. For sucrose Cotton devised the following tests: 10 c.c. of the sample are mixed with 0.5 gram of powdered ammonium molybdate, and Io c.c. of dilute hydrochloric acid (1 to 10) are added. In a second tube, 10 c.c. of pure milk or 10 c.c. of a 6% solution of lactose are similarly treated. The tubes are then placed in the water-bath and the temperature gradually raised to about 80°. If sucrose is present, the milk will become blue, while genuine milk or milk-sugar remains un- altered unless the temperature is raised to the _boiling-point. According to Cotton, the reaction is well marked in the presence of as little as 1 gram of sucrose to 1000 c.c. of the milk. For the detection of other organic thickening agents, such as pectoses, agar and mixtures of agar and gelatin, see under ‘‘Cream,’’ page 67. Calcium Saccharate (Saccharate of Lime).—A compound produced by the action of lime on sucrose has been used as a thickening agent. A test due to Bauer and Neumann is recommended by Lythgoe, from whose description the following is taken: To 25 c.c. of milk (or cream) add 10 c.c. of DETECTION OF ADULTERATION 47 5% solution of uranium acetate, shake well, al- low to stand for five minutes and filter. To 1o c.c. of the clear filtrate (in the case of cream use the total filtrate, which will beless than 10 c.c.) adda mixture of 2 c.c. saturated ammonium molyb- date and 8 c.c. dilute hydrochloric acid (1 part 25% acid and 7 parts water), and place in a water-bath at atemperature of 80° for five minutes. If the sample contains sugar the solution will have a prussian blue tint. This should always be compared in a colorimeter with the standard prussian blue solution prepared by adding a few drops of potassium ferrocyanid and 5 drops of 10% hydrochloric acid to a solution of 1 c.c. of 0.1% ferric chlorid in 20 c.c of water. It has been claimed that pure milk will give this test. Occasionally samples of pure milk will give a pale blue, but this can be entirely removed by filtration, and the filtrate will be green; while the color due to sucrose will pass through the filter, giving the blue solution characteristic of adulterated samples. The color is due to re- duction of molybdic acid, and is caused by levulose and dextrose as well as by sucrose. Solutions of 1 gram of lactose, levulose, dextrose and sucrose in 35 c.c of water were used in com- paring the amount of color produced when heated with the molybdenum reagent for five minutes. Lactose produced no color, levulose gave a heavy 48 MILK blue, sucrose a weaker blue and dextrose the weakest blue, corresponding in intensity as rio NM ae ie Stannous chlorid and ferrous sulfate give this color, but the reaction takes place in the cold, and with small quantities the color disappears on heating. In order for the color to persist after heating the sample of cream must contain these substances to the extent of 1% calculated as the metal. In this case the sample will be completely coagulated and the taste will be disagreeable. Hydrogen sulfid will also give the blue, but it will disappear on heating. If the solution does not show blue before heating, it is free from hydrogen sulfid, ferrous sulfate or stannous chlorid. As a confirmatory test for sugar, the resorcinol test may be applied to the serum prepared with uranium acetate as described. This test is given by sucrose and levulose, but not by dextrose or lactose. The quantitative estimation of sucrose in milk is given under Milk Products (page 74). Detection of Heated Milk.—Fresh milk con- tains one or more enzyms of the ‘‘peroxydase”’ type, that is, having power to bring about transfer of oxygen from peroxids to oxidable substances. As the function of these enzyms is destroyed by temperatures near 100°, it be- comes possible to utilize the reaction for deter- DETECTION OF ADULTERATION 49 mining whether a given sample has been thus heated. In most cases the action of the enzym is in- dicated by the production of a deep blue, no color change occurring when the enzym has been heated. Hydrogen peroxid is commonly em- ployed for furnishing the oxygen. A considerable number of substances have been found to be susceptible to oxidation under the influence of the milk enzyms. Benzene derivatives, com- monly used as photographic developers are especially susceptible. Guaiacum was first used. Arnold’s Method.—A solution of guaiacum in acetone is, according to Arnold and Menzel better than the ordinary tincture. The test is applied by adding to a small amount of the sample in a test-tube, about 10 drops of the guaiacum solution, to which a drop or two of hydrogen peroxid solution has just been added, so that the reagent will float on the milk. If the sample has not been heated above 80°, the point of contact of the liquids will show a deep blue ring. As guaiacum is liable to changes both in the solid form and in solution it is important to de- termine if the reagent is sensitive to raw milk, hence a control test should aways be made. Other reagents are now available which are, in the main, more trustworthy. Dupouy’s Method.,—In this method, 1-4 diam- inobenzene is used. The reagent is dissolved in 50 MILK water (a weak solution will suffice), a few drops added to the sample, then a few drops of hydrogen dioxid solution, and the liquids shaken gently. Milk that has not been heated above 80° gives immediately a bright blue. Milk that has been heated above this temperature shows no color change at first but may slowly acquire a bluish tint. This test is much in favor, but it is open to the objection that the solution of the reagent does not keep more than few hours, and even in the solid state some commercial samples soon decompose. Benzidin Method.—Wilkinson and Peters sug- gested this reagent, employing a solution of it with a few drops of acetic acid followed as usual by the oxidizing agent. Leffmann finds that the commercial benzidin hydrochlorid (furnished for volumetric estimation of sulfates) acts satis- factorily without acetic aicd. Wilkinson and Peters’ test is performed simi- larly to those just described, and has a similar significance. They give experiments to show that the method is rather more delicate than with diamino-benzene or guaiacum. ‘The solu- tion of the benzidin compound keeps better. They found that milk heated to 77° had lost its reactivity to guaiacum but retained reactivity to the other two reagents. Heated to 78° the reactivity was also lost to these. DETECTION OF ADULTERATION ee Leffmann has found that several commercial photographic developers, e. g., amidol, are ap- plicable in this test with about the limitations above noted. | At critical temperatures, however, the results with all the reagents depend materially on the length of the heating. Colors.—Annatto, turmeric, and some coal-tar colors are much used. Caramel is occasionally used, saffron and carotin but rarely. Annatto may be detected by rendering the sample slightly alkaline by acid sodium carbonate, im- mersing a slip of filter-paper, and allowing it to remain over night. Annatto will cause a reddish- yellow stain on the paper. Leys gives the following method for detecting annatto; 50 c.c. of the sample are shaken with vo"ee: Of ‘95 alcohol, so \c:e)),of ether) 3.0, af water, and 1.5 c.c. of ammonium hydroxid solution (sp. gr. o.goo), and allowed to stand for twenty minutes. Thelowerlayer, which in pres- ence of annatto will be greenish-yellow, is tapped off and gradually treated with half its measure of 10% solution of sodium sulfate, the separator being inverted without shaking, after each addi- tion. When the casein separates in flakes that gather at the surface, liquid is tapped off, strained through wire gauze, and placed in four test- tubes. To each of these amyl alcohol is added, 52 MILK and the tubes shaken and immersed in cold water, which is gradually raised to 80°. The emulsion breaks up, and the alcohol, holding the annatto in solution, comes to the surface. The alcoholic layer is separated from the lower stratum, evaporated to dryness, and the residue dissolved in warm water containing a little alcohol and ammonium hydroxid. Clean white cotton is introduced and the liquid evaporated nearly to dryness on the water-bath. The cotton, which is colored a pale yellow, even with pure milk, is washed and immersed in a solution of citric acid, when it will be immediately red- dened if the milk contains annatto. Saffron, turmeric, and the coloring-matter of the marigold do not give a similar reaction. Coal-tar colors may often be detected by dyeing wool, but Lythgoe has devised the following method, which is satisfactory: 15 c.c. of the sample are mixed in a porcelain basin with an equal volume of hydrochloric acid (sp. gr. 1.20), and the mass shaken gently so as to break the curd into coarse lumps. If the milk con- tains an azo-color, the curd will be pink; with normal milk the curd will be white or yellowish. General Method for Colors in Milk.—Leach devised a general method. 150 c.c. of the sample are coagulated in a porcelain basin, with the addition of acetic acid and heating, DETECTION OF ADULTERATION 53 and the curd separated from the whey. The curd will often collect in a mass; but if this does not occur, it must be freed from whey by straining through muslin. The curd is macerated for several hours in a closed flask, with occasional shaking, with ether to extract fat. Annatto will also be removed by it. The ether and curd are separated and treated as follows: The ether is evaporated, the residue mixed with a little weak solution of sodium hydroxid, and passed through a wet filter; and when this has drained, the fat is washed off and the paper dried. An orange tint shows annatto, which may be confirmed by a drop of solution of stannous chlorid, which makes a pink spot. If the curd is colorless, no foreign coloring- matter is in it; if orange or brown, it should be shaken with strong hydrochloric acid in a test- tube. If the mass turns blue gradually, caramel is probably present. The whey should be ex- amined for caramel (see page 95). If the mass turns pink at once, an azo-color is indicated. Falsification of the ‘‘Cream-line.’’—The use of glass bottles for retail delivery of milk enables purchasers to make approximate estimations of the richness of the sample by the depth of cream formed after standing for some time, this being 54 MILK of distinctly different tint from the milk below it. Deception has of late been extensively practised by a treatment of milk which breaks up the fat globules and increases the volume of cream formed, so that aslightly skimmed milk will yield afair volume ofcream. Determination of fat by the usual methods will show the fraud. See page 65. It has been found that many of the bottles used for distribution of milk are not of the capac- ity designated on them, but this is a matter of police regulation. Perservatives.—These are largely used, es- pecially in the warmer season, as a substitute for refrigeration. Many of them are sold under proprietary names which give no indication of their composition. Preparations of boric acid and borax were at one time the most frequent in use, but at present formalin, a 40% solution of formaldehyd, has come into favor. Sodium benzoate is now in common use as a preservative of cider, fruit-jellies, and similar articles, and may, therefore, be found in milk. Salicylic acid is not so much employed. Sodium car- bonate is occasionally used to prevent coagula- tion due to slight souring. Fluorids and abrastol may be used. A mixture of boric acid and borax is more efficient than either alone. The quantity generally used is equivalent to about 0.5 gram of DETECTION OF ADULTERATION 55 boric acid per tooo c.c. Formaldehyde is an efficient antiseptic. In the proportion of 0.125 gram to 1000 c.c., it will keep milk sweet for a week. Hydrogen peroxid, ozone and _ dichro- mates have been used. The almost universal decree of sanitary authorities is that milk must be free from any added material, but owing to its comparatively high cost, liability to decomposition and the marked characters of even incipient decomposition, great tempta- tion to use preservatives exists and any anti- septic, not actively poisonous, may be used. It has been.found that milk drawn and marketed under strict sanitary precautions will keep for a considerable time, even at moderate tempera- tures. The only permissible method of pre- serving milk is by refrigeration. In addition to the descriptions of the detec- tion and estimation of preservatives given below, see also under “‘Cream.”’ Formaldehyde. Hehner’s Test—Hehner found that when milk containing formaldehyde is mixed with sulfuric acid containing a trace of a ferric compound, a distinct blue appears. Richmond and Boseley showed that the delicacy of the test is much increased if the milk is diluted with an equal volume of water and sulfuric acid of 90 to 94%, added so that it forms a layer underneath the milk. Under 5 56 MILK these conditions, milk, in the absence of for- maldehyde, gives a slight greenish tinge at the junction of the two liquids, while a violet ring is formed when formaldehyde is present even in so small a quantity as 1 part in 200,000 of milk. The color is permanent for many hours. In the absence of formaldehyde, a brown ring may form in the course of a few hours, but it is below the junction line of the two liquids. Phenylhydrazin Test.—The following test avoids the fallacy of some other tests. A pinch of phenylhydrazin hydrochlorid is added to a few c.c. of the sample, the liquid shaken, then a drop of a fresh solution of sodium nitroprussid and a few drops of sodium hydroxid solution. A greenish tint is at once produced if formalde- hyde is present. If the test is applied to the liquid obtained by distilling milk the color will be deep blue. . Phloroglucol Test—A small amount of a 1% solution of phloroglucol is added to the sample and then a considerable volume of sodium hydroxid solution. In the presence of formalde- hyde a distinct rose tint will be produced. It is best to add the phloroglucol by means of a tube passed to the bottom of the test-tube. Bonnet’s test utilizes the vapor of formalde- hyde, and avoids the fallacies of some of. the DETECTION OF ADULTERATION be | older tests. A solution is made by dissolving 0.035 gram pure morphin sulfate in 10 c.c. of sulfuric acid. This solution does not keep well. A convenient amount of the sample is placed in a dish or beaker, a watch-glass containing 1 c.c. of the above solution is floated on it, and the dish covered with a glass plate. The materials are allowed to remain undisturbed at room-tempera- ture for several hours. Formaldehyde is in- dicated by the development of a color ranging from pink to dark blue. A black discoloration is disregarded. Bonnet found that with 1 part of formaldehyde to 25,000 parts of sample a distinct color appeared in one hour. In testing ice-cream and similar articles it must be borne in mind that some of the flavor- ing materials being aldehydic in nature may simulate formaldehyde. La Wall has found that vanillin may act thus. The phenylhydrazin and Bonnet tests are least liable to fallacy in this respect. Nitrites and Formaldehyde.— Mixtures of these substances are now sold under fanciful and mis- leading names, for milk preservatives as a nitrite prevents the reactions of formaldehyde with some of the tests. Leffmann has found that the phenylhydrazin test will react promptly with formaldehyde in presence of notable amount of nitrite and also 58 MILK that the well-known test for nitrites (sulfanilic acid and alphanaphthylamine) reacts in the presence of formaldehyde. The reactions are obtained in fresh samples and in those that have stood for twenty-four hours. Determination of Formaldehyde.—In the case of milk the proportion of formaldehyde is almost always small and it may be in great part removed from milk by distillation especially in a current of steam. B..H. Smith found that if 100) cc: of the sample are distilled with 1 c.c. of dilute sulfuric acid (1:3), one-third of the formaldehyde present will come over with the first 20 c.c. Distillation of milk is troublesome owing to bumping, but Smith found that it could be safely conducted with a flat evaporating burner. It is advisable to put a few pieces of pumice into the flask. Shrewsbury and Knapp recommend the fol- lowing method for estimation of formaldehyde. An oxidizing reagent is prepared by mixing o.1 gram of pure nitric acid (sp. gr. 1.52) with roo c.c. of strong hydrochloric acid are mixed. This mixture should be freshly made. 5 c.c. of milk are treated with 1o c.c. of the reagent, the mixture well shaken and kept for ten minutes in a water-bath at 50°. The depth of color is proportional to the amount of formalde- hyde present and by means of milk containing DETECTION OF ADULTERATION 59 known amounts of the preservative estimations may be made. Hydrogen Peroxid.—Many tests have been devised for detection of this substance. Among the most convenient and satisfactory is the reaction with vanadic acid first given by Werther. It may be carried out by adding to to c.c. of the milk, 10 drops of a 1% solution of vanadic acid in dilute sulfuric acid. This solution may be conveniently made by dissolved commercial sodium orthovanadate in the dilute acid. In the presence of hydrogen peroxid a distinct red will appear promptly. Barthel states that a proportion of o.o10 gram of the peroxid in 100 c.c. of milk can be detected positively using only 10 c.c. of the sample. Benzoates and Salicylates—The following method covers both these preservatives. 10 c.c. of dilute sulfuric acid (5%) are added to 20 c.c. of 95% alcohol and into this 50 c.c. of the milk are poured in a fine stream with constant stirring. After a few moments, the mixture is filtered, the filtrate being returned until it passes clear. A sufficient volume of the filtrate is extracted in the usual manner with an equal volume of ether or similar solvent. The solvent is divided into two portions that are separately evaporated and tested for benzoic and salicylic acids respectively as given below. 60 MILK Benzoates.—This is detected by a modification of Mohler’s method by Von der Heide and Jakob as given by U. S. Bureau of Chemistry. The residue that is to be tested for benzoic acid is dissolved in a little water, the solution mixed with from 1 to 3 c.c. of normal sodium hy- droxid and evaporated to dryness. To this resi- due is added from 5 to 1oc.c. of concentrated sul- furic acid and a small crystal of potassium nitrate and the mixture heated either for ten minutes in a glycerol bath between 120° and 130° or for twenty minutes in boiling water. If heated in the glycerol bath the temperature must not be permitted to go over 130°. Metadinitrobenzoic acid is formed. After cooling 1 c.c. of water is added, the liquid made decidedly ammoniacal, boiled to break up ammonium nitrite, and some fresh colorless ammonium sulfid solution added so that the liquids do not mix. A brown ring at junction indicates benzoic acid. The liquids being mixed, the color diffuses and on heating changes to greenish-yellow. The last reaction distinguishes benzoic acid from salicylic and cinnamic acid as these latter form amino-deriva- tives which are not destroyed by heating. Phenolphthalein interferes with this process. Salicylic Acid—The other portion of the ether-extract may be evaporated and tested for DETECTION OF ADULTERATION _ 61 salicylic acid in the usual manner with a ferric compound. Saccharin.—A suitable amount of the sample (so or 100 c.c.) is acidified with dilute (25%) sulfuric acid and extracted with a mixture of equal parts of petroleum spirit (boiling below 60°) and ether. The solvent is evaporated at a gentle heat. The presence of saccharin in the residue may’ be, detected by the taste.,..2 c:cy, of a saturated solution of sodium hydroxid are added and the dish heated until the residue dries and then to 210°—215°, and maintained thus for half anhour. The saccharin is converted into salicylic acid, which may be detected in the residue by acidulating it with sulfuric acid and applying the ferric chlorid test. If salicylic acid be present originally in the sample, the residue from the petroleum spirit and ether solution is dissolved in 50 c.c. of dilute hydrochloric acid, bromin water added in excess, the liquid shaken well, and filtered. Salicylic acid is completely removed as a brominated derivative. The filtrate is made strongly alkaline with sodium hydroxid, evapo- rated, and fused as described above. Sodium Carbonate and Sodium Acid Car- bonate.—These substances are occasionally added to milk to prevent acidity due to decomposition. Barthel recommends a test devised by Hilger. 50 c.c. of the milk are diluted with 250 c.c. of 62 MILK water, the mixture is heated, precipitated with a small amount of alcohol and a _ convenient volume filtered. The filtrate is evaporated to half its bulk. The presence of an alkali-carbon- ate is easily ascertained by the usual tests. Borates.—Jenkins’ method is convenient and reasonably delicate. 10 c.c. of milk are mixed with 7 c.c. of hydrochloric acid, filtered, a strip of turmeric paper dipped in the filtrate, and then dried on a watch-glass on the water-bath. The paper becomes red in the presence of borates. A simple test is to mix in a porcelain basin a drop or two of the milk, a drop of hydrochloric acid and a drop of alcoholic solution of turmeric and evaporate to dryness on the water-bath. The residue touched with ammonium hydroxid will show a distinct greenish stain in the presence of very small amounts of borates. It is obvious that the delicacy of both these tests may be materially increased by concen- trating the sample. As boric acid is volatile with steam it is best to render the sample slightly alkaline with sodium hydroxid before evaporating. Abrastol (Asaprol).—This is a calcium beta- naphthol-sulphonate that has marked antiseptic powers and has been used as a food preservative. The following test suggested by Leffmann will detect very small amounts. 10 c.c. of the sample are mixed with o.5 c.c. of the solution of DETECTION OF ADULTERATION 63 mercuric nitrate described on page 37. In the presence of abrastol a distinct yellow tint is produced in a few minutes. Greater delicacy can be obtained by using the same proportion of the reagent with 10 c.c. of milk known to be pure. Organic Contamination.—Sanitary control of market-milk also involves tests for animal prod- ucts, such as pus.cells, and the identification of specific microbes, such as those causing tuber- culosis and typhoid fever. These investiga- tions, however, are mostly outside of the scope of a work on chemical analysis. For informa- tion concerning these recourse must be had to works on pathology and bacteriology. Several chemical tests have been published by which it is claimed that approximate deter- mination of these contaminating organisms and substances can be made but they are not capable of replacing the exact methods of the pathologic and bacteriologic laboratory. One of these is the following. A dilute solution of methylene blue is prepared by adding 5 c.c. of a saturated alcoholic solution of the dye to 200 c.c. of water. 0.5 c.c. of this solution is added to 10 c.c. of the sample. If the color is discharged promptly, the sample contains over 100,000,000 bacteria per c.c. Hydrogen dioxid has been shown by the in- 64 MILK vestigations of Rentschler to kill quickly many forms of microbes, and may be applicable to the purification of milk, when, as in war, systematic protection and inspection are not possible. Preservation of Samples.—For the preservation of milk samples for a day or two, refrigeration is the best method. Sterilization in the ordinary steam sterilizer used in preparing culture-media, will enable milk to be kept for a considerable time if in a flask closed with a cotton plug. Several preservatives have been proposed for keepingsamples. Richmond found smallamounts of hydrofluoric acid effective, but it has been but little used. Formaldehyde is very efficient; in large amount it increases the total solids, inter- feres with the reactions of the proteins and simu- lates some of the reactions of the carbohydrates. A couple of drops of commercial formalin to 25 c.c. will preserve a sample for several days. MILK PRODUCTS CREAM Cream differs from whole milk principally in the fat-content; the analytic procedures, there- fore, follow those indicated under ‘‘ Milk,” except that the high fat may render some modifica- tions advisable. It is better, for instance, to weigh rather than measure cream, and it is often advisable to dilute it with a known weight of water. For the determination of fat the Rédse-Gottlieb method is much in favor (see page 72). The following are some special procedures. Imitation Cream.—By means of special ma- chinery, the fat globules of milk may be broken into very small portions without causing them to coalesce. This is termed ‘‘homogenizing’’ and will give to poor cream an appearance of richness. It is also possible to incorporate butter with skim-milk, producing an article resembling cream. Of course, unsalted, un- colored butter must be used. As butter made in the usual manner, always contains water, the 65 66 MILK PRODUCTS adulteration may be detected by the change in the refractive power of the serum as described on page 42. H. C. Lythgoe, who has investi- gated this question, finds that samples adulter- ated with butter will give a refraction below 36.0. Results may be confirmed by taking the ash of the sour serum. A large amount of the sample is taken (as the yield of serum is small), soured with a pure culture of lactic acid bacillus, or with a little sour milk, shaken in a bottle until the fat and curd have separated, the serum drawn off ‘and: the ash’ of? 25) ‘c.c) taken) 1s should not be below 0.73%. The homogizing of cream without the addition of fat can be detected by microscopic examination. Formic Acid—Revis and Bolton state that glucose containing this may be found in cream and give the following method for its detection. 100 grams are diluted with an equal weight of water, 20 c.c. of a 20% solution of phosphoric acid added, and 1oo c.c. distilled, the end of the condenser dipping below the surface of milk of lime containing at least 1 gram of calcium hydroxid and 2 c.c. of 3% acetic acid, free from formic. The distillate is evaporated to dryness, sealed in a small tube of hard glass, drawn out at one end that dips into a small U-tube containing 2 c.c. of water, arranged so that none of the water can be drawn into the tube, and heated until CREAM 67 distillation ceases. The water in the U-tube is mixed with 2 c.c. of Schiff’s reagent. If formic acid was present, the mixture will become violet within a half hour. Schiff’s reagent is obtained by dissolving 1 gram of rosanilin hydrochlorid in Io c.c. of water, adding a mixture of 2 C.C. saturated solution of sodium acid sulfite and o.5 c.c. strong hydrochloric acid, then water to make 100 C.C. The solution keeps for some time in the dark. Agar.—This is now often used as a thicken- ing agent. Although characteristic diatoms are found in it, the detection of the substance by isolation of these has not been practically successful. Revis and Bolton recommend the following method. so grams of the sample are diluted with 100 c.c. of water, heated in boiling water and cleared with s cc. of 10% calcium chlorid solution. The mixture is filtered, preferably in a hot-water funnel, cooled and mixed with about two-thirds its volume of strong alcohol. The precipitate (containing any agar that may have been in the sample) is separated, and boiled with 5 c.c. of water until dissolved. If it contains agar, the solution will gelatinize on cooling. To detect the presence of gelatin in association with agar, the procedure is the same, except that when the precipitate is dissolved, a few c.c. of the solution 68 MILK PRODUCTS are treated with picric acid solution. A pre- cipitate indicates gelatin. In this case, the re- mainder of the solution is evaporated to small bulk, and mixed with a 10% solution of tannin until no more precipitate is produced. The liquid must in this treatment not have a tem- perature of over 60°. To it a few c.c of white of egg are added and the mixture heated to boiling for thirty minutes, filtered hot, concentrated to small bulk on the water and allowed to cool and gelatinize. CONDENSED MILK Commercial condensed milks present two prin- cipal forms, sweetened and unsweetened. In the latter sucrose is generally used. Often consti- tuting more than half the solids of the product. Up to recent years, unsweetened condensed milk was largely sold in the United States as “‘evapo- rated cream’’ but this is now forbidden by the federal food law and by many State enactments. Dried milk has also been manufactured but does not seem to have met with much favorable reception. Commercial evaporation of milk is conducted at a low temperature so that less modification of the ingredients is produced than in ordinary boiling, but some modification of the lactose may occur which will make polarimetric readings less accurate than with unheated milk. The analysis of unsweetened condensed milk can be conducted along the same lines as those for ordinary milk and cream, the sample being diluted about three times by adding a known volume of water. It must not be forgotten, that lactose may crystallize from condensed and dried milks, and excessive polarimetric rotation occur in recently made dilutions, unless these are heated to brief boiling and cooled (see page 39). Com- 69 70 MILK PRODUCTS mercial condensed milks usually represent whole milk concentrated to about one-third or two- sevenths of its original volume. A small amount of invert-sugar may be present. The most com- mon defect in condensed milks is deficiency in fat, due to preparation from closely skimmed milks. Preservatives (other than sucrose) and coloring-matters are rarely used, nor is it likely that foreign fats will be present. The fat of unsweetened condensed milk can be readily determined by the L-B method (page 18). In arecent publication, Bigelow andFitzgerald give the following detailed description of the application of the Leffmann and Beam method to the examination of unsweetened condensed milk: Weigh g grams of evaporated milk into an 8% Babcock milk bottle. Add 1oc.c. of water. Thoroughly mix by shaking and add 3 c.c. of a mixture of equal parts of amyl alcohol and con- centrated hydrochloric acid. Shake thoroughly and add 10 c.c. of concentrated sulfuric acid (1.84 sp. gr.) in three or four portions, mixing after each addition. If too much heat develops the bottle may be cooled somewhat in water during the addition of the acid. Fill the bottle to near the base of the neck with a hot fresh mixture of equal parts of sulfuric acid and water. Thoroughly mix the contents CONDENSED MILK 71 of the bottle by shaking. Raise the fat column to the top of the scale by means of the acid and water mixture, and whirl for five minutes. Read promptly (see page 20) from the extreme bottom of the fat column to the bottom of the upper meniscus. Multiply the reading by 2, and deduct 0.25; the remainder is the per cent. of fat. If an electric centrifuge without heat has been employed, the fat column will be somewhat cool and should be heated, before reading, in a water- bath about 60°. The same authors give the opinion that the centrifugal methods are not sufficiently accurate to be depended upon for determining if evapo- rated milk is up to standard. The Rése-Gottlieb method is best for this purpose. If the centrif- ugal methods are employed, considerable allow- ance must be made for inaccuracies. Results obtained are inaccurate unless the fat column is clear, with the meniscus at the bottom of the column perfect and not distorted by either char or milky appearance. The percentage of solids as calculated from the sp. gr. is not sufficiently accurate to determine whether the milk complies with the standard unless the correction factor for the for- mula of calculation is ascertained frequently by the determination of solids by drying. 6 72 MILK PRODUCTS The full analysis of sweetened condensed milk is difficult, and many of the published figures are erroneous. ‘The sucrose interferes with the ex- traction of the fat by solvents. The same difficulty occurs in the analysis of some prepared infant-foods, such as mixtures of milk with malt and glucose. For the general operations, a portion of the well-mixed contents of a freshly opened can Should be accurately weighed, diluted with a known amount of water, and well mixed, from which mass the portions for analysis may be taken and the results calculated to the original sample. 50 grams mixed with 150 c.c. of water will be a convenient quantity. For the polar- imetric determination of lactose, a special pro- cedure will be necessary; but for determination of solids, ash, total proteins, and total reducing sugars, the examination may be made as with ordinary milk upon this diluted sample. Fat.—The Adams method is not satisfactory under ordinary conditions, owing to the sucrose. The Rése-Gottlieb method is now largely used and generally approved. The following descrip- tion is given by Bigelow and Fitzgerald: Weigh from 4.5 to 5.0 grams evaporated or condensed milk into a Rése-Gottlieb tube, add water to make about 11 grams and 14% to1 CONDENSED MILK 73 c.c. concentrated ammonium hydroxid, and thor- oughly mix by shaking. Add toc.c. 95 % alcohol and shake thoroughly. Fill up to the level of the side tube with water, if necessary, and shake. Add 25 c.c. ether and shake wellfor oneminute. Add 25c.c. petroleum spirit (b. p. below 65°) and shake well for one minute. Allow tube to stand until layers separate well. Draw off ether-fat solution ascompletely as practicable and run it through a small quick- acting filter into a weighted flask (weighted by counterpoising, if the test is not finished the day it is started.) Re-extract the liquid in tube as before with 15 c.c. of each of petroleum spirit and ether, shaking after eachisadded. Before the addition a little alcohol may be added and the contents of the tube mixed by shaking, to bring the layer of ammoniacal liquid close up to the outlet tube, for by repeated extractions the surface of sepa- ration is lowered. Run the solution from the second extraction through the filter into the flask, and wash end of spigot, filter paper, and lower surface of the funnel with ether; or, better, with a mixture of equal parts of ether and petroleum spirit which has been allowed to stand for separation of water. 74 MILK PRODUCTS In the examination of cream a third extraction is necessary, but with evaporated and condensed milk the third extraction does not recover more than 0.02 or 0.03 % of fat, and may be omitted. Evaporate the liquid slowly on a steam bath and dry the fat in steam oven until its weight is constant. Weigh after one hourand then at half- hour intervals. Assoon as the fat begins to gain in weight stop drying and take the next previous weight. Increase of weight is due to oxidation after all moisture and alcohol are gone. In all cases the drying should be completed the day it is begun. Double Extraction.—The following is given as a provisional A. O. A. C. method: Extract with ether, as usual, about 5 grams of a 40% solution, dry, leave the tubes in a dish containing at least 500 c.c. of water, dry, extract again with ether for four hours. Sugars.—If regard is to be given to the presence of invert-sugar, a special method must be followed. The processes first given consider lactose and sucrose only. The heating employed in the manufacture of condensed milk may reduce the rotatory power of lactose sufficiently to cause error in the polarimetric method. ‘The reducing power with alkaline copper solutions is not seriously affected. Determination of sucrose may be made by CONDENSED MILK 75 difference; that is, subtracting the sum of the other ingredients from the total solids. This will serve for ordinary inspection purposes, since the amount present is almost always large, gener- ally more than the total of milk-solids, and a slight error does not affect the judgment as to the wholesomeness of the sample. Exact work requires, however, that the sucrose be de- termined directly. Several processes have been devised for the purpose. Sucrose exerts but little action on Fehling’s solution, but invert- sugar acts powerfully, and some processes depend on determining the reducing power before and after inversion. Since the polarimetric reading is also markedly changed by the inversion, the difference in polarization may be employed. Fermentation may be so conducted as to re- move the sucrose (also any form of glucose) while the lactose is unaffected. This method is chiefly valuable for recognizing invert-sugar or either of its constituents. Inversion Methods.—These must be such as to secure prompt inversion of the sucrose without affecting the lactose. Experiment shows that citric acid and invertase are the most suitable agents. Stokes & Bodmer have worked out the citric acid method substantially as follows: 25 c.c. of the diluted sample are coagulated by addition of 1% of citric acid, without heating, 76 MILK PRODUCTS and made up to 200 c.c. plus the volume of the precipitated fat and proteins (see page 38). The liquid portion, which now measures 200 C.c., is passed through a dry filter. The reducing power with alkaline copper solutions is determined at once upon 50 c.c. of this filtrate. To another so c.c., 1% of citric acid is added, the solution boiled at least thirty minutes, and the reducing power also determined. ‘The increase over that of the first solution is due to the invert-sugar formed by the action of the citric acid on the sucrose. It is necessary to bear in mind that the reducing equivalents of lactose and invert-sugar are not the same. Volumetric methods may be employed. The following method is based on the difference in polarimetric reading before and after action of invertase. 75 c.c. of the diluted milk are placed in a too-c.c. flask, diluted to about 80 c.c., heated to boiling, to correct birotation, cooled, and ro ¢.c. of acid mercuric nitrate) solunen (page 37) added. ‘The mixture is made up to 100 c.c., well shaken, filtered through a dry filter, and the polarimetric reading taken at once. It will be the sum of the effect of the two sugars. ‘The volume of the sugar-containing liquid is calcu- lated by allowing for the precipitated proteins and fat, as described on page 38. so c.c. of the filtrate are placed in a flask CONDENSED MILK 77 marked at 55 c.c., a piece of litmus paper dropped in, and the excess of nitric acid cautiously neu- tralized by sodium hydroxid solution. The liquid is then faintly acidified by a single drop of acetic acid (it must not be alkaline), a few drops of an alcoholic solution of thymol are added, and then 2 c.c. of a solution of invertase, prepared by grinding half a cake of ordinary compressed yeast with 1o c.c. of water and filtering. The flask is corked and allowed to remain at a tem- perature of 35° to 40° for twenty-four hours. The cane-sugar will be inverted, while the milk-sugar will be unaffected. The flask is filled to the mark (55 c.c.) with washed aluminum hydroxid and water, mixed, filtered, and the polarimetric reading taken. The amount of cane-sugar can be determined from the difference in the two readings by the formula ii tooa +b t 142.68 — ia in which S is the percentage of sucrose; a, the reading before, 06, after inversion; ?, the temperature. LACTOSE, SUCROSE AND INVERT-SUGAR.—Bige- low and McElroy propose the following routine method to include invert-sugar. The reagents are: 78 MILK PRODUCTS Acid Mercuric Iodid.—Mercuric chlorid, 1.35 grams; potassium iodid, 3.32 grams; glacial acetic acid, 2 c.c.; water, 64 C.c. Alumina-cream.—A cold saturated solution of alum is divided into two unequal portions, a slight excess of ammonium hydroxid is added to the larger portion and the remainder added until a faintly acid reaction to litmus is obtained. The entire contents of the can are transferred to a porcelain dish and thoroughly mixed. A number of portions of about 25 grams are weighed carefully in 100 c.c. flasks. Water is added to two of the portions, and the solutions boiled. The flasks are then cooled, clarified by means of a small amount of the acid mercuric iodid and alumina cream, made up to mark, filtered, and the polarimetric reading noted. Other portions of the milk are heated in the water-bath to 55°; one-half of a cake of com- pressed yeast is added to each flask and the temperature maintained at 55° for five hours. Acid mercuric iodid and alumina-cream are then added, the solution cooled to room tem- perature, made up to mark, mixed, filtered, and polarized. The amount of sucrose is determined by formula given above. Correction for the volume of precipitated solids may be made by the double-dilution method. The total reducing sugar is estimated in one of the portions by one of CONDENSED MILK 70 the reducing methods, and if the sum of it and the amount of sucrose obtained by inversion is equal to that obtained by the direct reading of both sugars before inversion, no invert-sugar is present. If the amount of reducing sugar seems to be too great, the lactose must be re-determined as follows: 250 grams of the condensed milk are dissolved in water, the solution boiled, cooled to 80°, a solution of about 4 grams of glacial phos- phoric acid added, the mixture kept at 80° for a few minutes, then cooled to room temperature, made up to mark, shaken, and filtered. It may be assumed that the volume of the precipitate is equal to that obtained by mercuric iodid solu- tion. Enough sodium hydroxid is then added to not quite neutralize the free acid, and sufficient water to make up for the volume of the solids precipitated by the phosphoricacid. The mixture is then filtered and the filtrate is measured in portions of 100 c.c. into 2o00-c.c. flasks. A solution containing 20 milligrams of potassium fluorid and half a cake of compressed yeast is added to each flask, and the mixture allowed to stand for ten days at a temperature between 25° and 30°. Invert-sugar and sucrose are fermented and removed by the yeast in the presence of a fluorid; lactose is unaffected. The flasks are filled to the mark and the lactose determined either by reducing or by the polariscope. The amount 80 MILK PRODUCTS of copper solution reduced by the lactose and invert-sugar, less the equivalent of lactose re- maining after fermentation, is due to invert- sugar. BUTTER Butter is a mixture of fat, water, and curd. The water contains lactose and the salts of the milk. Common salt is usually present, being added after the churning. Artificial coloring is frequently used. Butter-fat is distinguished from other animal fats in that it contains a notable proportion of acid radicles with a small number of carbon atoms. Thus, about 91% consists of palmitin and olein and the remainder of butyrin and caproin, along with small amounts of caprylin, caprin, myristin, and some others. According to the experiments of Hehner & Mitchell, stearin is present only in very small quantity. The exact arrangement of the constituents is unknown. The composition of good commercial butter usually ranges within the following limits: eB ee Us citer te tah ia ae aN Re aot 78% to 94% rere FA er TR Me SY bee ORE 1% to 3% hGH Fe ROMY EA PEON ER eh tee Oe Fe CEM ere se eee 5% to 14% Yad cal GR ee ea ta er le mk 0% to 7% Butter containing over 40% of water is some- times sold. Such samples are pale and spongy, lose weight, and become rancid rapidly. 81 82 MILK PRODUCTS The official methods of the A.O.A.C. for the analysis of butter are as follows: Preparation of the Sample.—If large quantities of butter are to be sampled, a butter trier or sampler may be used. The portions thus drawn, about 500 grams, are to be perfectly melted in a closed vessel at as low a temperature as possible, and when melted the whole is to be shaken vio- lently for some minutes until the mass is homo- geneous and sufficiently solidified to prevent the separation of the water and fat.