Determination of Ammonia and Urea in Milk - American Chemical

of ammonia by the Berg and Sherman (1) method showed re- markably good ... adaptations of the Folin and Bell (J) Permutit procedure origi- nally desig...
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FEBRUARY 15, 1938

ANALYTICAL EDITION

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hexavalent selenium by sulfur dioxide.) The selenium was weighed as usual. c.4DMIUM DETERMINATION. A 0.1000-gram sample was disCd 58.74% 8e 4 1 . 2 6 % solved in nitric acid in an Erlenmeyer flask equipped with a boiling valve to trap the spray. The excess acid was removed 100.00% by evaporat’ion, and the residue was fumed with 1 cc. of sulfuric acid. The resulting cadmium sulfate was then dissolved in This fractional precipitation m a y be repeated several times. about 5 cc. of water, and saturated sulfurous acid solution wag I n one experiment, three successive precipitations yielded added in successive small portions with intermittent warming, until selenium no longer precipitated. The solution was then precipitates, the average purity of which was 97.29 per cent. EFFECT OF LIGHTUPON THE COLOROF THE PRECIPITATE. filtered and heated gently t o expel most of the sulfur dioxide. The last trace of sulfur dioxide was oxidized with potassium Exposure t o light darkens the color of the precipitate. Thus, permanganate, which also served to oxidize any remaining solutions mixed i n the dark i n the stoichiometric ratio protraces of selenious acid to selenic acid. (The latter compound is less readily reduced at the cathode during the electrolysis which duced a white t,o canary-yellow precipitate of lo^ b u t varifollows.) able selenium content, whereas the precipitate formed by A slight excess of oxalic acid was now added to reduce the mixing the solutions in ordinary daylight was bright red in excess potassium permanganate, and the solution was neutralcolor. T h e light yellow tints were stable in the dark for ized with sodium hydroxide, acidified with 1 cc. of acetic acid, diluted to nearly 100 cc., and electrolyzed between 50” and 70” C., hours, but a few seconds in direct sunlight sufficed t o redden using a platinum gauze cathode. (By beginning the electrolysis the exposed granules of the precipitate. Qualhative exiyith the smallest voltage that will cause an appreciable current periments with various filters indicated that radiat>ionin the to flow, say 0.05 ampere, and after an hour increasing the curvisible range was most effective. Quantitative studies, rent to 0.10 ampere, a smooth deposit, free from loose crystals, was obt,ained.) however, have not yet been made. After 3 or 4 hours a few cubic centimeters of water were added, and if no fresh deposit formed above the former solution level Analytical on the cathode, the electrolysis was interrupted, taking the utmost, care lest the cadmium deposit be redissolved by the The selenium content of SODIUM SELETOSULFATE SOLUTION. electrolyte after the cessation of the current. The cathode was the sodium selenosulfate solution was determined by adding a thoroughly mashed in distilled water. follon-ed by alcohol and large excess of hydrochloric acid and weighing the precipitated ether, and dried in an oven at 100’ for 5 minutes or until the selenium. odor of ether was no longer perceptible. The gain in weight of Digestion with aqua regia was SELENIUM DETERMINATIOS, the cathode was the weight of cadmium in the sample. unsatisfactory, as low, inconsistent results were obtained. Excellent results JTere obtained by digesting the sample with Literature Cited a small quantity of sodium peroxide in about 5 cc. of water until all dark particles were converted into a white, gelatinous mass. (1) Bassett and Durant, J. Chem. Soc., 1927, 1401. (For a 0.1000-gram sample of cadmium selenide it is convenient 1 2 ) hlellor. J. W.. “Comprehensive Treatise on Inorganic and Theoto use 1 cc. of 30 per cent hydrogen peroxide and 0.5 gram of retical Chemistry,” Vols. IV and X, Sew York, Longmans, sodium hydroxide in a volume of about, 5 cc.) A large excess Green & Co., 1923. of oxidant is to he avoided, because of evolution of chlorine in ( 3 ) O’Brien, W. J., U. S. Patent 1,894,931(assigned to Glidden Co.). subsequent operations. An excess of hydrochloric acid was ‘4) Rathke, J . prakt. Chem., 95, 1 (1865). added, and sulfur dioxide was bubbled through the solution ~ 5 Vortmann, ) Ber., 22, 2307 (1589). until the selenium precipitate was coagulated. (The solution should contain at least, 90 per cent by volume of concentrated RECEIVED November 16, 1937. Contribution from the Chemical Laborahydrochloric acid, in order to facilitate complete reduction of tories of Johns Hopkins Univer-ity and Central College.

The theoretical composition is

Determination of Ammonia and Urea in Milk A. E. PERKINS, Ohio .4gricultural Experiment Station, Wooster, Ohio

I

N CONNECTIOS mith another investigation it was necessary for t h e writer t o make determinations of ammonia in the milk produced b y differently fed groups of cows. After unsuccessful experience with several of the methods found in the literature, a n accurate yet simple method was devised which more nearly met t h e requirements. A method has also been devised for the accurate deterinination of urea in milk, using chemicals and equipment found in most laboratories.

Determination of Ammonia The ammonia content of milk has been a matter of interest a n d study for a t least 80 years. for Bouchardt and Quevenne in 1857 ( 2 ) are reported to have observed a n ammonia content of 0.193 per cent in the case of milk made alkaline with sodium hydroxide before distillation. Of course this high result for ammonia was due to a breaking down of other nitrogenous compounds and did not represent a true value for ammonia or ammonium salts in the milk. Modern historv with respect to this determination may be said to begin wiih the work of Berg and Sherman ( 1 ) and of

Sherman anti several collaborators ( 4 , 1.7, IS). hlilk itself was mixed with an equal volume of neutral methyl alcohol, using sodium carbonate as an alkali, and distilled under partial vacuum in a very large flask to overcome the pronounced tendency to foam. Values observed for fresh market milk from S e w York City and vicinity averaged around 0.39 mg. per 100 cc. Milk either untreated or preserved with formalin and stored 8 to 14 days shcwed values for ammonia up t,o 20 mg. p w 100 cc. Tillmans, Splittgerber, and Iiiffart ( I 6) reported a series of results obtained bv their own method of first precipitating the ammonia as ammonium magnesium phosphate from proteinfree milk serum. After filtering and washing, this precipitate was distilled in the presence of alkali. Parallel determinations of ammonia by the Berg and Sherman ( I ) method showed remarkably good agreement. Other groups of workers, notably .Kieferle and Gloetzl ( S ) , Burstein and Frum (3),and Kluge (D), have made different adaptations of the Folin and Hell (;) Permutit procedure originally designed for the determination of ammonia in urine. Recently, Siemrzycki and Gerhardt (1I ) , Polonovsky and Boulanger j I J ) , and other groups of workers hare reported the use of a combination of steam and vacuum distillation carried o u t on an aqueous deproteinized milk filtrate in an apparatus devised originally hg Parnas and Heller (I?), for the determination of ammonia in blood. This method would seem capable of arcurate result$, h i t the apparatus seems too complicated and limited in it.s application to make its general use a t all pruhahle.

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INDUSTRIAL AND ENGINEERING CHEMISTRY

PREVIOUS METHODS.The methods reviewed would seem to fall into two definite classes: (1) those using some form of distillation technic for separating ammonia from other milk components, and (2) those depending on some adaptation of the Folin and Bell (7) Permutit technic to accomplish this purpose. Values for ammonia in fresh milk obtained by methods of the first class fall in the range of 0.3 to 0.4 mg. per 100 cc. of milk, and the results are reasonably uniform (1, 4, i i , 1 4 , 16, 18). TABLE I. DEVELOPMENT OF AMMOZ;IA IN MILK ON AGING (Milligrams of ammonia nitrogen in 100 cc. of milk) Fresh 24 Hours 4 Days 6 Days 8 Days 1 2 Day3 Samples in Laboratory Window Raw 0.24 0.42 0.38 0.90 2.25 5,6 Pasteurized 0.35 0.42 0.43 0.48 0.48 5.0 Fresh 48 Hours 7 Days 14 Days 21 Days 30 Days Samples in Cooler a t 5' C. 0.30 0.42 1.55 5.63 11.0 Raw 0.28 Pasteurized 0.34 0.50 0.48 1.00 7.2 0.32

The results obtained by methods of the second class, using the Folin and Bell (7) Permutit adsorption principle, are decidedly different and more variable than those quoted above. Kieferle and Gloetzl (8) report ammonia values of 0.7 to 1.6 mg. per 100 cc. for the ammonia nitrogen content of fresh milk by this procedure. The writer in attempting to follow their method has demonstrated to his own satisfaction, however, that the high ammonia values reported by Kieferle and Gloetel are due to a breaking down of urea during the heat precipitation of the lactalbumin in acetic acid solution. This explanation seemed evident because samples of milk of high urea content were affected to a much greater extent than others of low urea content, and samples of pure urea subjected to the same treatment gave high values for ammonia. Burstein and Frum (3) and Kluge (9), using the Permutit adsorption principle, report ammonia values for fresh milk of around 0.1 to 0.2 mg. per 100 cc. or even lower in some cases. Burstein and Frum treat the milk itself directly with Permutit, while Kluge conducts the ammonia adsorption on the serum from trichloracetic acid precipitation of the milk. Burstein and Frum do not mention recovery determinations, while Kluge claims satisfactory recoveries. The writer has repeatedly attempted to make determinations of ammonia in milk by the adsorption procedure, using both of the above and other modifications and has been unable to obtain what were considered to be satisfactory or consistent results. Recovery determinations of added ammonia were most unsatisfactory. The tentative conclusion was reached that the adsorption of ammonia on Permutit is unsatisfactory when applied to milk, probably because some material in the milk or milk serum inhibits satisfactory ammonia adsorption. h h K IXocuTABLE11. AMMONIACONTEXTOF PASTEURIZED LATED WITH PURE CULTURES OF VARIOUS B.4CTERIAL SPECIES Ammonia Ammonia Time Nitrogen Time Nitrogen Hours MgJ% Hours Ma./% Samples Held a t Temperatures between 10' and 18' C. ~treptococcuslactis 76 1.75 220 9.92 76 1.16 220 4.06 Streptococcus liquefaciens 76 1.50 220 4.30 Aerobacter aerooenes 76 0.90 220 1.33 Bacillus subtilis 220 1.25 Escherichia coli 76 0.87 76 1.08 220 1.25 Lactobacillus bulgaricus 76 1.40 220 2.23 Patudomonas jraoi i6 0.80 220 1,12 B1. pasteurized milk Samples Held at Laboratory Temperature, about 26' C. 27 7.60 52 9.12 Streptococcus lactis 2: 1.30 52 1.72 Sireptococcua liquejaciens 27 3.30 52 5.37 Aerobacter aerogenes 28 0.90 52 1.15 Bacillua aubtilis 28 0.35 54 0.60 Eacherichia coli 28 1.08 54 2.00 Lactobacillus bulgaricus 28 1.28 54 2.53 Paeudomonas fragi 28 0.35 54 2.50 BI. pasteurized milk Name of Culture

VOL. 10, NO. 2

Some of the distillation methods quoted above depend on a low boiling point obtained by means of alcoholic solutions and distillation in partial vacuum to prevent the breaking down of the other nitrogenous substances to ammonia during the distillation of the latter. Other methods precipitate and remove these substances as far as possible before distilling. I n the method described below the writer has sought to combine both these advantages. PROPOSED METHOD.Kieferle and Gloetzl(8) made a comparison of various methods of milk protein precipitation. One of the most complete precipitants in the list studied was magnesium sulfate in combination with alcohol. However, Kieferle and Gloetzl made no use of this method, in connection with the determination of ammonia or urea. The filtrate from this precipitation has been used by the writer as a suitable medium in which to carry out both ammonia and urea determinations. The alcoholic magnesium sulfate filtrate contains only about 30 nig. per 100 cc. of nitrogen based on the volume of the milk taken, whereas the milk itself contains from 400 to 600 or more mg. of nitrogen per 100 cc.; thus about 92 to 95 per cent of the nitrogen is removed from the scene of the reaction. The alcoholic filtrate boils about 20" C. below the boiling point of m t e r , lessening the danger of decomposing the remaining 5 to 8 per cent of nonprotein nitrogen. Data preJented in Table I11 show that the remaining nitrogenous constituents of this protein-free milk filtrate are little affected under the conditions of the distillation. Several samples have been distilled under a vacuum of 550 mm. The results in comparison with those obtained with the usual procedure were not appreciably affected by this added precaution. TABLE111. EFFECTOF OTHERNONPROTEIN NITROQEX INGREDIENTS ON AMMONIA DETERMINATION (Pure materials distilled as in the ammonia. determination) Amount Ammonia Material Used Produced Mg. MQ

.

The procedure found effective and convenient for ammonia determination in milk is as follows: One hundred cubic centimeters of milk are treated with 20 grams of anhydrous magnesium sulfate. Alcohol of 85 to 95 per cent concentration is then added, with one intermediate shaking, to a final volume of 500 cc. The material is then filtered through a paper filter after standing a short time. The volume of filtrate which separates spontaneously may be increased to about 430 cc. by enclosing the paper filter in cloth and pressing by hand. A 200-cc. portion of the above alcoholic filtrate representing 40 cc. of milk is transferred to a 500-cc. Kjeldahl flask and treated with 0.5 t o 1 gram of magnesium oxide. Boiling is continued in the regular Kjeldahl nitrogen apparatus until 125 to 150 cc. of distillate are recovered, the ammonia being received in a solution of 0.00714 N sulfuric acid. The excess acid is then titrated with 0.00714 N ammonia, using a very dilute and carefully neutralized solution of methyl red as indicator. After deducting the proper hlank value each cubic centimeter of 0.00714 N acid used represents 0.1 mg. of ammonia nitrogen.

DISCUSSIOK.The method calls only for standard equipment and chemicals available in most laboratories and is very economical of time. The blank due to reagents is very small and the recovery of added ammonia very good, as shown in Table IV. Increasing the alkalinity of the mixture at the time of precipitation by the addition of magnesium oxide or calcium hydroxide appeared to increase the recovery of added ammonia but it also increased the blank determination, as also shown in Table IV.

FEBRUARY 1.5, 1938

ANALYTICAL EDITION

The amount of sample and reagents specified allows for distillation in duplicate. The amount of alcohol used may seem excessive, but most of this can be readily recovered by distillation from the acidified solution. The better grades of methyl alcohol or ethyl alcohol denatured with methyl would doubtless also be suitable where they are more readily available than ethyl alcohol. Some of the results obtained by the use of this method are shown in Tables I to V. TABLEIV. DETERWHATION OF Amror;ra Recoveries

IN

MILE

Recovery of Added Ammonia 2 mg per 10 mg. per 100 cc. added 100 cc. added

%

%

69-77 93-96 89-100

70 83 90

Blanks AIgSOr

MgSOh

MgSOh

+ alcohol + N g O + alcohol + Ca(OH)* + alcohol

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that from others fed on high-protein rations. Apparently the intake level of protein does not affect the ammonia-nitrogen content of milk.

Determination of Urea The presence in milk of considerable amounts of urea hay been recognized by numerous workers during the past 20 years. Denis and Minot (5) suggested the treatment of milk directly with the Marshall ( I O ) urease extract in determining its urea content, the milk so treated being later aspirated by the Van Slyke and Cullen (17) procedure for the removal of the ammonia formed. The ammonia was then determined, either by titration or nesslerization. The Denis and Minot method ha. heen follon ed by other workers TABLE VI.

TOTAL NITROGEN IN 111LK P R O D U C E D O V D I F F E R E V T FEEDING LEVELSOF PROTEIN

0.02 0.10

Lou-Protein. SutritiTe Ratio 1 13

0.17

0,002

Xumerous claims have been made regarding the advantages of ammonia determination as a means of sanitary control (3, 9). Other than showing that ammonia development occurs chiefly as an accompaniment of prolonged bacterial action, no attempt is macle in this article to confirm or deny such claims. Many questions arise regarding the effect of different feeds, various production practices, differences in the condition of the cow, or different ways of processing or storing on the ammonia content of the milk. For most of these questions the available data are not sufficient to warrant very specific statements. Formerly it was believed that contamination with stable air and various impurities was largely responsible for ammonia in milk. Burstein and Frum (3) studied these factors but found no definite effect on the ammonia content of the milk. Ordinary commercial pasteurization has little effect on the ammonia content of the fresh milk, but may have a marked effect on the subsequent course of ammonia development, as shown in Table I. It will also be observed from this table that ammonia developed very slowly. The increase was not at all marked until the sample showed other evidences of souring. Table I1 shows the same slow development of ammonia for samples of pasteurized milk inoculated with pure cultures of various bacterial species when the samples were kept at relatively low temperatures. When samples of pasteurized milk were similarly inoculated but held at laboratory temperature, the ammonia production was greater in 27 hours than in 76 hours a t the lower temperature (Table 11). It is evident that the various bacterial species differ greatly in both rapidity and total capacity of ammonia production. T'4BLE V. AMMOSIAX-ITROGENCONTENT OF MILKB Y A U T H O R ' S METHOD (Milligrams in 100 cc. of milk) 4 Cons, 21 Samples Highest Louest 0 60 0 27 Lla-protein feeding 3 Cows, 20 Samples Highest Lowest 0 55 0 23 High-protein feeding

Mean 0.42 Mean 0.41

Other determinations of ammonia and of titratable acidity a t regular intervals after inoculation seemed t o show a possible relationship between these two values. The presentation of details regarding these points, however, is outside the scope of the present work. I n Table V is given the ammonia nitrogen content of milk from cows fed on low-protein rations in comparison with

Total nitrogen Casein nitrogen Albumin nitrogen, tannic acid precipitation Residual or nonprotein nitrogen rrea

Normal

High-Protein, Nutritive Ratio 1.2

%

%

%

100 78.2

100 75.4

100 70.4

19.1 2.7 0.7

20.6 4.3 1.9

20.8 8.8 4.5

I n attempting to carry out urea determinations by this general procedure, the writer has found it preferable to use the filtrate remaining after the removal of casein or that remaining after the removal of both casein and albumin rather than the milk itself, because of the pronounced tendency of the milk proteins to break down with the formation of ammonia under the influence of even the mild alkalies used in the aeration. I n the use of such deproteinized filtrates, however, care must usually be taken to avoid a n excess of protein precipitant in the solution which is treated with urease, since many of the precipitants destroy or inhibit the action of this enzyme. The alcoholic magnesium sulfate filtrate described above, however, is favorable for the action of urease.

TABLE mr. cow

No. of Determinations

293 301 362

10 9

3

329 332 414

3 9 5

UREA NITROGES IN MILK

Maximum

,np./roo

cc.

Minimum M o . / l O O cc.

Average

M'a./roo

Low-Protein Feeding 8.0 5.2 7.2 1.7 9.2 1.6 Av. of 22 determinations High-Protein Feeding 27.4 20.8 22.4 17.5 28.7 20.7 Av. or' 17 determinationa

cc.

6.2 3.5 3.6 3.9 24.3 20.3 25.0 22.4

DETERMINATION. A convenient amount of the alcoholic magnesium filtrate described above (20 cc., equivalent to 4 cc. of milk) is used. One-half as much of a 5 per cent extract of Jack Bean meal in 25 per cent alcohol is added and the mixture is diluted to 200 cc., incubated for 2 hours at 40" C., or allowed t o stand overnight at room temperature. One gram of magnesium oxide is then added and the distillation and titration of the resulting ammonia are carried out as directed above. After deducting the amount of preformed ammonia nitrogen from the observed value, the remainder may be considered as urea nitrogen or converted to urea by use of the factor 2.214. Other principles of urea determination are available, but Van Slyke and Cullen (17) have shown that the urease decomposition is relatively complete and altogether a simple and reliable procedure. Complete aspiration of the ammonia by the Van Slyke and Cullen procedure requires approximately 2 hours, whereas the distillation of ammonia as described in the present paper can usually be carried out in 10 minutes. Commercial urease preparations may be used in place of the Jack Bean extract, if desired.

INDUSTRIAL AND ENGINEERING CHEMISTRY

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DISCUSSION.Data presented in Table \‘I, calculated from work previously published by the writer ( I S ) , show that urea nitrogen constitutes about one-half of the nonprotein nitrogen found in average milk. The proportion is larger in the case of milk from cows heavily fed on protein and is decidedly smaller in the milk from cows fed rations deficient in protein. Denis and Minot (6) showed an apparent increase in the urea content of milk from the use of high-protein feeds. Data obtained by the writer and presented briefly in Table VI1 show that the urea content of milk is affected t o a remarkable extent by the level of protein feeding. According to the author’s observations (13) it is the only one of the nonprotein nitrogenous constituents of milk affected to any very marked extent by variations in the amount of protein fed. Acknowledgment The author is indebted to H. H. Weiser, Ohio State Cniversity, for the pure cultures of bacteria described in Table 11.

Bouchardt and Quevenne, Du Lait, Paris [Quoted by Tillmans, Splittgerber, and Riffart (IS)], 15 (185’7). (3) Burstein, A. I., and Frum, F. S., Z. Untersuch. Lebensm., 69,

(2)

5, 421 (1936). (4)

Colwell, R. H., and Sherman, H. C., J. Bid. Chem., 5, 247

11908). --, (5) Denis, W., and hlinot, A. (6) Ibid., 38,453 (1919). \--

S.,Ibid., 37, 353

(1919).

(7) Folin, 0.. and Bell, R. D., Ibid., 29, 329 (1917). (8) Kieferle, F., and Gloetal, J., Milchw. Forsch., 11, 62 (1931). (9) Kluge, H., 2. Untersuch. Lebensm., 71, 232 (1936). (10) Marshall, E. K., Jr., J . Bid. Chem.. 14, 283 (1913). (11) Niemcayoki. S., and Gerhardt, K., Lait, 16, 1049 (1936). (1’2) Parnas, J. K., and Heller, J., Conzpt rend. SOC. bid., 91, 706 (1924); Biochem. Z., 152, 1-28 (1924). (13) Perkins, A. E., Ohio Agr. Exgt. Sta., Bull. 515 (1932,. (14) Polonovsky, M., and Boulanger, P., Bull. S O C . chim. bid., 17,

G., J Bml. Chem., 3, 171 (1907) (1U) Tillmans, J , Splittgerber, A , , and Rlffart, H., Z Untersuch. ,Yahr u. Genussm., 27, 59 (1914). (17) Tan Slyke, D. D., and Cullen, G E., J . Bid. Chem., 19, 211 119141.

(18) Whitman,

W.G.. and Sherman, H. C., J. Am. Chem. Soc., 30,

1288 (1908).

Literature Cited (1) Berg, IT. K., and Sherman, H. C., (1905).

VOL. 10, NO. 2

1937. Presented before the Division of Agricultural Chemistry at the 94th Meeting of the -4merican Chemical Society, Rochester, N. Y . , September 6 to 10, 1937. RECEIVEDOctober 8,

S.A m . Chern. Soc..

27, 124

and Food

Determination of the Equivalent Acidity and Basicitv of Fertilizers J

A Study of Mixed Indicators W. H. PIERRE, NELSON TULLY, AND H. V. ASHBURN West Virginia -4gricultural Experiment Station, Morgantown, W. Va.

I

N T H E method for determining the equivalent acidity and basicity of fertilizers (9))the acid solution of the fertilizer is titrated by means of methyl red to an end point corresponding to the neutralization of the first hydrogen of phosphoric acid. That methyl red gives the desired end point was shown by the fact that the equivalent basicity values of mono-, di-, and tricalcium phosphates were found to agree with the theoretical values. IYith most solutions the end point of methyl red (first change in color from reddish pink to slightly orange pink) is easy to note, provided the titration is carried out under proper light conditions and a blank is used for comparison. Since most mixed fertilizers contain considerable amounts of phosphorus, however, the solution titrated is highly buffered and the color change per unit of base added is not as large as is ordinarily obtained in analytical procedures. The tendency among various workers, therefore. especially when inexperienced with the determination, is to titrate past the end point. hforeover, with solutions containing colloidal precipitates of iron and aluminum phosphate the appearance of turhidity may be mistaken for a change in color. Horat ( b ) proposed the use of bromophenol blue instead of methyl red. Like all simple indicators, however, the transition interval of bromophenol blue extends over a range of about 1.0 p H unit, and it is difficult to depcribe the exact color change a t the desired end point. Nethpl red and bromophenol blue were compared by a number of collaborators of the .issociation of Official Agricultural Chemists, but there was no agreement as to which indicator was more satisfactory.

The ideal indicator for use in the determination of the equivalent acidity and basicity of fertilizers would give (1) the end point a t the proper pH value, (2) a definite color change in both clear and turbid solutions, (3) a color change that can be easily described so that various workers will titrate to the same end point, and (4) warning of the approaching end point. During recent years a greater amount of attention has been given to “achromatic” or other mixed indicators (6). Since

FIGURE 1.

TITRaTION CTJRVES OF PHOSPHORIC ACID AND OF A C I D EXTRACTS OF V.4RIOUS FERTILIZER6