an improved method for the estimation of inorganic phosphoric acid in

AN IMPROVED METHOD FOR THE ESTIMATION OF INORGANIC PHOSPHORIC ACID IN CERTAIN

TISSUES AND FOOD PRODUCTS.’

BY ROBERT M. CHAPIN AND WILMER C. POWICK. (From the Biochemic Division, Bureau of Animal Industry, U. S. Department

of Agriculture, Washington.)

(Received for publication, December 12, 1914.)

INTRODUCTION.

For the solution of the much discussed questions as to the precise significance of the organically and of the inorganically combined phosphorus in animal and in vegetable tissues, a method for sharply and surely differentiating between these two classes of compounds and for estimating accurately the amount of phosphorus combined in each is obviously essential. With a view to supplying this need, many methods, differing among themselves in accuracy and in practicability, and each more or less specifi- cally adapted to the investigation of a certain ‘limited range of materials, have been evolved; but as yet no method has been found, if indeed one ever can be found, that is universally superior in its application to all substances. The improved method here offered, however,-designed for and successfully used in the determination of inorganic phosphorus in eggs and in meats,-is believed to possess a sufficient number of points of superiority over its immediate competitors, together with a sufficiently wide range of applica- bility, to justify its publication at this time.

DESCRIPTIVE PART.

Discussim of improvements claimed for the method.

Of the methods now available for the determination of inorganic phosphorus in flesh, there are three which seem to stand pre-

1 Published by permission of the Secretary of Agriculture. 97

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98 Estimation of Inorganic Phosphoric Acid

eminent; viz., the Emmett and Grindley method,2 the Siegfried and Singewald method,3 and the Forbes methodd-ach of which has undoubtedly proved reasonably satisfactory in its particular field. Yet their failure to guard sufficiently well against tedious filtrations, decomposition of organic phosphorus compounds, and incomplete separation of the organic from the inorganic phosphorus, makes problematical the accuracy and practicability of each when applied to tissues other than flesh, or even to that tissue after the occurrence of degenerative changes.

The requirements of a generally trustworthy method for the determination of inorganic phosphorus in physiological tissues, the shortcomings of the above mentioned methods, and the advantages claimed for the improved method are briefly summarized below.

Extraction. To insure the solution of any water-insoluble phosphates that might be present, the extracting medium should be acid. To avoid any possible chemical decomposition of the organically combined phosphorus, the use of strong reagents and of heat should be avoided. As a precaution against bacterial and enzymatic changes, the extraction should be effected quickly, in the cold, and in the presence of an antiseptic. Most simply to prevent the interference of colloids in the subsequent phosphate precipitation and the unnecessary exposure of the organic phosphorus compounds to the action of the precipitant, these substances should be rendered initially insoluble in the extracting medium, either by physical or by chemical methods. For obvious reasons, time-consuming operations should be avoided as far as possible.

The Emmett and Grindley method falls short of the above requirements by the use of a neutral solvent and of heat; the Sieg- fried and Singewald method, by failure to remove the protective colloids, by exposure of the organic phosphorus to the action of the phosphate precipitant, and by the long duration of the initiil

2 A. D. Emmett and H. S. Grindley: Jour. Am. Chem. Sot., xxviii, p. 25, 1906; H. S. Grindley and E. L. Ross: this Journal, viii, p. 483, 1910.

aPI. Siegfried and E. Singewa,ld: Ztschr. f. Untersuch. d. Nahrungs u. Genussmittel, x, p. 521, 1905.

4 E. B. Forbes, A. Lehmann, R. C. Collison, and A. C. Whittier: Bull. 215, Ohio Agric. Exper. &a., 1910.


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R. M. Chapin and W. C. Powick

filtration; and the Forbes method, by the use of heat and the neces- sity for a double filtration. Finally, Collison’s5 modification of the Forbes method of extraction is open to the criticism that the proteid superficially coagulated by the strong alcohol interferes with the further penetration of the tissue by the solvent.

In the method herein proposed, however, the above requirements have been met by employing as the extracting medium an aqueous solution of picric acid containing a small amount of hydrochloric acid. The extraction is complete; bacterial action is prevented and the proteids are effectually coagulated by the reagent; while the danger of chemical or enzymatic changes is mini- mized by the low temperature and the rapidity of the extraction. The extract is easily filtered and is practically free from organically combined phosphorus; while by the use of an aliquot of the filtrate for further work, a tedious washing of the precipitate is avoided.

Separation of the organic from the inorganic phosphorus. In view of the meagerness of our knowledge regarding t.he organic phosphorus compounds in physiological tissues, the seijaja&n of these from the inorganic phosphates must be conducted largeIy according to a priori considerations. Upon such grounds it would appear that the best assurance of a quantitative separation of the inorganic from the organic phosphorus and from other contami: nating substances is to be found in an at least partial*separa&n at the time of the extraction (which we have secured by the use of picric acid) and a subsequent double precipitation of the inorganic phosphates, once from acid and once from alkaline solution. Obviously, the precipitant first to be employed should be that which is least likely to decompose the organic phosphorus compounds, or, by precipitating them, to expose them to decomposition in subsequent operations; while the final precipitant should be that which yields the precipitate best adapted for the estimation of small amounts of phosphoric acid.

Since, in the initial use of ammonium molybdate,-the only available acid precipitant,-the presence of strong reagents and the precipitation of more or less of the organic matter are in- volved, and since in its use as a final precipitant a compara- tively heavy precipitate is obtained from a relatively small amount

5 R. C. Collison: Jour. Ind. and Eng. Chem., iv, p. 666, 1912.


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LOO Estimation of Inorganic Phosphoric Acid

of phosphoric acid, the initial use of an alkaline precipitant is clearly indicated. Of these, magnesia mixture seems to be pref- erable to either barium chloride or calcium chloride, for by its use a clear filtrate is easily obtained and the use of sulphuric acid in subsequent operations is not rendered impossible.

In the method here proposed, therefore, the initial precipitation has been made by means of magnesia mixture and the -final precipitation by means of ammonium molybdate, which, we believe, insures for the method a further superiority over the Emmett and Grindley method, in which the order is reversed.

Final estimation of the inorganic phosphoric acid. Unques- tionably the inorganic phosphorus should be determined directly and not obtained by difference; for it is the form of phosphorus concerning which the most precise information is usually desired, and it is likewise the form which is most amenable to exact determination.

There has been much study, resulting in a voluminous literature, upon the. final estimation of phosphoric acid; but as a criticism or comparison of the methods from time to time proposed does not lie~&hin the scope of this paper, the reader is referred to the werk df-Artmatm,‘j which contains a critical review of the methods now available. - The present writers have chosen to make their &al esthation gravimetrically, by the direct weighing of the ammonium phosphomolybdate: first, because the previous pro- &dqrb ti their method leads most logically (as has already been pointed-c&) to this method of final estimation; and secondly, which is more important, because the precipitate of ammonium phosphomolybdate is much larger (nineteen times heavier) than a chemically equivalent precipitate of magnesium pyrophosphate, an important consideration in view of the small amount of phosphoric acid usually dealt with.

The particular method of estimation that we have chosen is that of Lorenz,’ which has been used with satisfaction by a number of workers, including especially Neubauer and Liicker,* who have proved its reliability and have suggested slight modifications in its technique in the interests of convenience and economy.

6 P. Artmann: Ztschr. Angew. Chem., xxvi, pt. I, p. 203, 1913. 7 N. V. Lorenz: Die landwirtschajt. Versuchsst., Iv, p. 183, 1901; &err.

them. Ztg., xiv, p. 1, 1911. * H. Neubauer and F. Lticker: Ztschr. j. anal. Chem., li, p. 161, 1912.


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R. M. Chapin and W. C. Powick 101

Detailed execution of the method.

Extraction. Rejecting the method of extracting the phosphoric acid by exhausting the tissue by repeated washings, we have chosen, in the interests of convenience and simplicity and in order to se- cure a more concentrated extract, to subject the material to a single, thorough maceration with the sdvent, and to employ an aliquot of the filtrate for further work. We have devised three methods by which to determine the exact concentration of the extract in terms of the original material, thereby avoiding errors due to the presence of an originally unknown volume of water or of insoluble matter in the sample. These methods are described below as Modifications A, B, and C.

Moclijication A. This modification is applicable when the water content of the sample is known or can be determined with sufficient accuracy.

A weighed sample of the material, containing between 8 and 80 mgm. of inorganic PZOS, is macerated in a porcelain mortar with 20 grams of dry, acid-washed sand, and is thoroughly, but quickly, mixed with an accurately measured amount of water (about 200 cc.) and 10 cc. of 2.5 normal hydrochloric acid. The mixture is transferred to a wide mouthed, glass-stoppered bottle of about 500 cc. capacity, and 5 to 8 grams of powdered picric acid are added. The bottle is shaken at frequent intervals during the next two hours, or continuously, by machine, for one-half hour; after which the extract is filtered through a folded filter, and 100 cc. of the filtrate are measured out for subsequent work.

The volume of the whole solution is found by adding to the volume of water and hydrochloric acid employed the volume of water contained in the sample; and the proportional amount of the original sample represented by 100 cc. of the filtrate is then calculated.

Modijication B. This modification is applicable when the volume of the insoluble matter is known or is negligible.

The weighed material is ground with sand in a mortar, as under Modification A. The whole is then diluted and quantitatively transferred with water to a graduated flask or stoppered cylinder, where it is treated with 10 cc. of 2.5 normal hydrochloric acid and 5 to 8 grams of powdered picric acid as before.. It is then


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diluted with water to a definite to&l volume, and shaken, filtered; and sampled, as described under Modification A.

To determine the volume displaced by the insoluble matter, the residue is sucked dry, its weight and specific gravity are determined, and its volume is calculated. If the use of sand is unnecessary and the amount of insoluble matter is negligible, obviously the volume correction may be omitted; while if sand is employed, but the volume of the insoluble matter is negligible, only a once for all determined correction for the volume of the sand need be applied, provided, however, that in each case the picric acid is added after the apparent volume of the solution has been observed.

ModiJicution C. In this case a “marker” is used to indicate the concentration of the resultant extract, on the principle that if a definite amount of some inert chemical that is not naturally present in the material under examination be added at the begin- ning, a determination of the concentration of such chemical in the final extract will afford a measure of the total volume of liquid in which this chemical, and therefore the P205, is dissolved. A half normal solution of potassium iodide has been chosen for this purpose, and the urea-nitrite method of Schirmerg has been used for the determination of the potassium iodide in the filtered extract and for the standardization of the half normal potassium iodide solution. The method of Kendall,lO which was first tried, proved to be inapplicable in the presence of picric acid, while the other methods of Schirmer were less suited to our purpose than the method chosen.

In this modification the weighed material is ground with sand as under Modification A, and by the aid of water is quantitatively washed into a 500 cc. glass-stoppered bottle and brought to a volume of about 200 cc. The mixture is then treated successively with 10 cc. of 2.5 normal hydrochloric acid, exactly 25 cc. of the half normal potassium iodide solution, and from 5 to 8 grams of powdered picric acid. The bottle is shaken and its contents filtered, as described under Modification A; and from the filtrate two portions of 50 cc. and 100 cc. are measured for the potassium iodide and for the phosphoric acid determinations, respectively.

g W. Schirmer: Arch. d. Pharm., ccl, p. 448, 1912. lo E. C. Kendall: Jour. Am. Chem. Sot., xxxiv, p. 894, 1912.


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R. M. Chapin and W. C. Powiek 103

The 50 cc. portion is measured into a spacious, narrow necked, glass-stoppered bottle, treated with 10 cc. of five normal sulphuric acid and 15 cc. of a freshly prepared 1 per cent sodium nitrite solution, and after one to two minutes with 1.5 grams of crystal- lized urea. The solution is shaken vigorously in the tightly stoppered bottle until the excess of nitrous acid is destroyed, after which the separated iodine is dissolved by the addition of 10 cc. of normal potassium iodide solution and titrated in the usual manner against thiosulphate in the presence of starch. The thiosulphate equivalent of 5 cc. of the original half normal potassium iodide solution is determined in the same manner, and multiplied by five to obtain the thiosulphate equivalent of the potassium iodide originally added to the sample. If this figure be represented by A cc., and the thiosulphate required for the 50 cc. aliquot of the filtrate by B cc., and the weight of the tissue operated upon by W gm., then the 100 cc. of the filtrate to be used for the PZ05 determination will obviously represent “F grams of the original material.

Separation of the inorganic phosphoric acid. The phosphoric acid in the 100 cc. aliquot of the filtrate obtained by either of t,he above modifications of the extraction process is precipitated in the usual manner with magnesia mixture and ammonia;. and the precipitation is completed, or allowed to complete itself, by agi- tation or by sufficient standing. The precipitate is filtered off and washed with 2.5 per cent ammonia water until the washings are practically colorless; then the precipitate on the filter and in the beaker is dissolved in dilute nitric acid, and washed with water into a 150 cc. beaker. The combined solution and washings are then evaporated to dryness and the residue is redissolved in a mixture of 25 cc. of nitric acid (sp. gr., 1.20), 1 cc. of concentrated sulphuric acid, and 25 cc. of water.‘l

Final estimation of the inorganic phosphoric acid. The final. estimation of the inorganic phosphoric acid in the nitric-sulphuric acid solution of the dry residue, prepared as above, we have carried out by the Lorenz method, with exact adherence to the directions which he has given, both for the precipitation

11 Ammoniacal alcohol might, of course, be used for the washing of the magnesium ammonium phosphate precipitate and acidified alcohol for dissolving it, according to the method of Forbes; but this procedure has not appeared to be necessary in our work thus far.


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and for the filtration and washing, except that in some cases we .have used acetone for the washing, as suggested by Neubauer and Liicker, in place of alcohol and ether, as directed by Lorenz. The use of acetone for this purpose we have found to be perfectly satisfactory, provided only that the acetone is free from aldehydes and is otherwise pure.

Discussion and explanation of the various steps of this method are to be found in the original references, and for the sake of brevity will be omitted here.

EXPERIMENTAL PART.

Experimental results in the development and application of the improved method.

Lorenz method. Chemically pure sodium biphosphate was dissolved in water, and the solution was standardized by weighing both the residue obtained by the evaporation of 100 cc. in a platinum dish, and the residue of sodium metaphosphate subse- quently obtained on ignition. Duplicate determinations all led to identical values for the concentration of the solution.

The P206 factor of the precipitate yielded by the Lorenz method was determined by weighing the precipitates obtained from varying quantities of the above solution, conditions in duplicate determinations being purposely varied between the limits speci- fied by Lorenz without appreciable irregularities resulting. The method was found to be thoroughly satisfactory, and the PZOs factors determined by us differed from those given by Lorenz by no more than the experimental error, as shown in the following table :

TABLE 1.

PzO, factors for use with the Lorenz method.:

gm. 1.5 1.0 0.8 0.4 0.3 0.12

Found by us

0.03307 0.03305 0.03298 0.03312

0.03322

Given by Lmenr

0.03291 0.03295 0.03299


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Originally the factors obtained by us for small amounts of PzOh were appreciably lower than those shown in the table, but this was found to be due to a separation of MoOa that had taken place in consequence of an insufficient amount of nitric acid in the reagent. In such work as was done with this reagent, however, the factors experimentally found with that solution were used. Lorenz has recommended the general use of the factor 0.03295, which, being based upon more exhaustive work than that done by us, we are inclined to accept in preference to our slightly different figures, particularly as the difference may be due to our having used a less pure salt.

TABLE 2.

Recovery of PzO6 from pick acid solution.

1 2 3 / 4 _______

P,Os used, gm.. . . . . . . . . _ . . . . . . . 0.004106 0.004106 0.004106 0.006159 Weight of precipitate, gm.. . . . 0.1257 0.1265 0.1257 0.1888 Factor.......................... 0.03259 0.03259 0.03259 0.03259 P206 recovered, gm.. . . . . . . . 0.004097 0.004113 0.004097 0.006153 PZOS recovered, per cent.. . . . . . 99.77 100.11 99.77 99.90

_______ PtOa recovered, average per cent. (.x . . . . . . . . . . .,99.89

E$ect of pi& acid 012 the recovery of Pz06. Measured portions of the standard phosphate solution were diluted to 100 cc. with a saturated solution of picric acid, and precipitated with magnesia mixture and ammonia. After twelve hours the precipitates were filtered off and washed, dissolved in dilute nitric acid, and the phosphoric acid was determined by the method already indicated. The results are given in Table 2.

Considering the small volume of the standard phosphate solution that was used, the results shown in Table 2 are extremely satisfactory.

Recovery of P205 added to eggs and to meat. In the next set of experiments a considerable quantity of whole egg substance was thoroughly mixed, and its water content was determined by drying a sample in a vacuum oven at about 65°C. Inorganic phosphoric acid determinations were then made upon portions of the original egg substance and likewise upon portions of the egg


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substance to which known amounts of the standard phosphate solution had been added, with a view to ascertaining how much of the added phosphoric acid could be recovered. The determinations were carried out by t,he method already described, the picric acid extract being made by Modification A. The results of this experiment are given in Table 3.

In another similar experiment on meat (see Table 6) in which the extracts were made by Modification C, 98.73 per cent, 101.12 per cent, and 98.97 per cent were recovered in triplicate determinations, making an average recovery of 99.61 per cent.

The difference between the amounts of phosphoric acid added and the amount recovered in the above experiments is in each case small; and since the loss or gain is divided between two separate determinations, each of which, in the case of the experiment on meat, involves an iodine as well as a phosphoric acid determination, the errors appear to be well within the permissible limits.

Experiments on the iodine method for ascertaining the concentration of the extract. The first step in this series of experiments was to check the Schirmer method for the estimation of potassium iodide.

A standard solution of potassium iodide was prepared from a weighed amount of the pure substance, and was checked by titration .against thiosulphate by Kendall’s method. The standard solution of thiosulphate had been based upon standard dichromate in the usual way. It was calculated that 1 cc. of standard potassium iodide solution should be equivalent to 013288 cc. of the standard thiosulphate. The results of the tests on Schirmer’s method, both in the presence and in the absence of picric acid, are given in Table 4.

The Schirmer method having been shown to be satisfactorily accurate, the whole method of PzOs determination according to Modification C was then carried out on a standard phosphate solution as follows :

To 10 cc. of a standard phosphate solution, containing a total of 0.0154 of a gram of P206, were added 25 cc. of a standard potassium iodide solution. To this mixture were added 10 cc. of a 2.5 normal hydrochloric acid solution, 5 grams of picric acid, and sufficient water to make the volume about 250 cc. Of the final solution two aliquot portions of 25 cc. were used for the


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TABL

E 3.

Reco

very

of

a kn

own

amou

nt

of Pz

Oa

adde

d to

eggs

.

I A

i PZ

Ol1

1

0.00

_.

0.

0102

7 0.

00

0.00

40.0

0 40

.00

40.0

0 73

.25

74.3

4 74

.34

29.3

29

.74

29.7

4

141.

1 10

.0

150.

09

10.0

15

0.09

10

.0

180.

4 18

9.83

18

9.83

0.30

52

0.03

296

0.01

0058

0.

0181

45

0.00

8087

0.11

62

0.03

259

0.09

3787

0.

0071

89

0.11

68

0.03

259

0.00

3807

0.

0072

26

0.06

7207

0.01

0058

97

.98

-. -. 98

.15

1 2

3 ~~

0.00

0.

00

0.00

2046

0.

0020

46

4.98

9 4.

989

0.01

0207

0.

0102

07

155.

08

10.0

.94.

82

194.

82

0.27

50

0 03

2834

0.

0090

29

0.01

7591

0.

0072

07

0.27

45

0.03

2834

0.

0090

13

0.01

7559

0.

0072

07

0.01

0352

10

1.42

0.00

2046

5.

017

Gm

. Pz

Oa

per

cc.

stan

dard

so

lutio

n ...

......

....

Cc.

adde

d ...

......

......

......

......

......

.....

Wei

ght

of a

dded

Pz

Os

......

......

......

......

...

Wei

ght

of

egg

subs

tanc

e,

gm .

......

......

......

. P

er

cent

of

m

oist

ure

......

......

......

......

....

Vol

ume

of

wat

er

in

char

ge.

cc.,

......

......

....

Vol

ume

of w

ater

+

Pa06

sol

utio

n.

oc .

......

....

Vol

ume

2.5

N

HC

I ...

......

......

......

......

..

Tota

l vo

lum

e of

sol

utio

n ...

......

......

......

..

Wei

ght

of

prec

ipita

te

from

10

0 cc

....

......

....

PZO

S fa

ctor

...

......

......

......

......

......

...

Wei

ght

of

PzO

a in

10

0 cc

....

......

......

......

. W

eigh

t of

PlO

a in

w

hole

ch

arge

...

......

......

. Av

erag

e Pa

Os

in

egg

used

....

......

......

......

.

Diff

eren

ce:

adde

d Pz

Os

reco

vere

d ...

......

.....

Per

cent

of

add

ed

PzO

s re

cove

red

......

......

...

Aver

age

per

cent

of

rec

over

y ...

......

......

...

0.00

*

0.00

0.

0102

7

I- .- - .- .- .- -

.- . .- .- ._

._

_.

-

40.0

0 73

.25

40.0

9 73

.25

.I- 1

29.3

144.

2 10

.0

183.

5 A

0.13

62

0.03

259

0.00

443!

0.

0081

4!

83.5

/

183.

5 18

0.4

I

_-

0.13

34

0.13

61

0.03

259

0.03

259

O.O

lM34

8 0.

0044

35

0.00

7978

0.

0081

39

0.00

8087

-A

0.30

58

0.03

296

0.01

0078

0.

0181

80

0.00

8087

0.01

0093

98

.32


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TABLE 4.

Results on standard potassium iodide obtained with Schirmer’s urea-nitrite method.

KI solution used, cc ............... Thiosulphate required, cc ..........

Average titration ................. Calculated titration ...............

iodine determination, and two portions of 50 cc. for the phosphoric acid determination by the combined magnesia mixture and Lorenz methods. The results are given in Table 5.

It remained finally to check the accuracy of Modification C in the presence of proteid, for which purpose the following experiment was carried out.

From a thoroughly ground sample of beef round, six portions of about 20 gm. each were accurately weighed into large porcelain mortars containing about 20 gm. of acid-washed sand. These samples were then each ground, with the addition of a little water, to a smooth paste, transferred quantitatively, with the aid of about 200 co. of water, to a glass-stoppered bottle of about 500 cc. capacity, and treated successively with 10 cc. of 2.5 normal hydrochloric acid, 5 gm. of picric acid, and 25 cc. of half normal

TABLE 5.

Application of the whole method (ModiJication C) to standard PzO6 solution.

1 2

Thiosulphate required for whole amount of KI used.. . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . . . . 16.50 cc. 96.50 cc.

Thiosulphate required for 25 cc. aliquot . . . . . 9.75 cc. 9.74 cc. Average.................................... 9.745 cc.

Per cent of whole solution represented by 25 cc. 10.098 Per cent of whole solution represented by 50 cc. 20.196

Weight of precipitate from 50 cc.. . . . . . . . . . . . Weight of PZOS recovered from 50 cc. (average). Weight of PZOS recovered from whole solution

(EW). . . . . . . . . . . . . . . . . . . . . . . . . . . . Weight of P206 added.. . . . . . . . . . . . . . . . . . . Per cent of added PZOS recovered.. . . . . . . . . . .

0.0962 gm. 0.0978 gm. 0.003162 gm.

0.01566 gm. 0.01564 gm.

100.11


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potassium iodide solution. To three of the samples, 25 cc. portions of a solution containing 0.001564 gm. of PzOs per cc. were added, and the inorganic phosphoric acid in each sample was then determined by Modification C of the method that we have already described. The results of this experiment are shown in Table 6.

TABLE 6.

Application of Modification C lo recovery of phosphoric acid added. to beef.

1

KI solution used, cc.. . . . . . . 25 .OO Thiosulphate equivalent of above 110.80 Volume of extract wed for iodine

determination.. . . . . . . 50.00 Tbiosulphate required for 50 cc... 19.96 Part of whole represented by

5occ . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1801~ Part of whole solution repre-

sented by 100 cc . . . . . . . . . . 0.3602

Charge........................... 20.1618 Gm. of meat represented by 100

cc. sliquot.. . . . . . . . . . . . . 7.2639 Weight of phosphomolybdate pre- .

mp1tate......................... 0.6253 PzOsfmtor....................... 0.0330

Weight of PzOs in 100 cc. (total). 0.0206‘ Per cent of “natural” PzO5 in

meat........................... 0.2841

Average of first set.. . .

2

25.00 110.80

50.00 20.08

0.181

0.362

20.031

7.260

0.621 0.033

0.020

0.282

--

23

146 -- 6

17

0 01 -. 50

3 1

25.00 25 .OO 110.80 110.80

50.00 50.00 20.35 19.70

.

0.18366 0.1778

0.367321 0.3556

20.0445 19.9806

7.3627 7.0980

0.6421 1.0270 0.03301 0.0330

~___ 0.02119 0.0339

0.2878

0.2847

Weight of “nr&mtl” PtOs in 100 cc ............................... ( 0.0202

Weight of added PzOa determined in 100 cc ....................... 0.0137

Pa06 originally added ............................................ 0.0391l Added PzOs recovered (calculated for wholej .................... 0.0386

Per cent of added P*Os recovered ................................. 98.73

- --

--

4

4

1

3

0 1

I 1

2

25.00 110.80

50.00 20.21

0.1824

0 3648

20.0095

7.2995

1.0654 0.0330/

0.03521

0.0207S

0 01442

0.0391( 0.03954

101.12

!I-

I -

Average percentage of recovery . . . . . . ,. . . . . . . . . . . . . . . . . . .

25.00 JO.80

50.00 22.96

0 20722

0.41444

20.3118

8.4180

1.2097 0.03307

0.04ooo

0.02396

0.01604

0.03910 0 03870

98.97

99.61 --

While the averages of the individual experiments described above, both in the control and in the experiment on meat, show the practically quantitative recovery of the entire amaunt of phosphoric acid added, yet the recovery was not so nearly quantitative in the individual determinations, which show errors amounting to over 1 per cent of the total phosphoric acid determined. When it is considered, however, that the actual experi-


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mental error arising from a combination of four separate analytical operations is naturally considerable, and that in these experiments such an error is multiplied by three, a final error of * 1 per cent does not seem inordinately large.

It may therefore be said that, in the absence of interfering substances, the above described modification leads to rapid and sufficiehtly accurate results, and for the most part seems to be a very desirable variation. Yet it is felt that the modification has not yet received a sufficiently broad test, and that it leaves something to be desired on account of the possibly limited range of its utility. Thus, it was found that it could not be used upon cold water extracts of meat which had been preserved with thymol, on account of the interfering action of that substance, unless the thymol had been ‘previously expelled.

Comparison of the improved method with other well known methods.

Although the data already obtained had convinced us of the accuracy of our method, it was nevertheless decided to compare it with a few other well known methods, even though in the event of their yielding discrepant results, no conclusion could be formed as to which method might be at fault. The methods selected for this purpose were the Emmett-Grindley, the Siegfried-Singe- wald, and the Forbes methods. These methods as outlined by Grindley and Ross*2 were followed in all essential respects, except that after the initial phosphomolybdate precipitates had been formed according to directions, the subsequent procedure in all the methods was identical and consisted of a magnesia mixture precipitation followed by a final estimation of the phosphoric acid by the Lorenz method.

The material selected as sample was a cold water extract of flesh, prepared with thymol as a preservative;’ and for the purpose of comparing the methods, the two following experiments were made.

Experiment A. Two liters of a cold water extract were prepared from practically fresh, finely ground beef round and water saturated with thymol. Triplicate determinations were made by each of the three methods

I2 Grindley and Ross : Zoc. cit.


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R. M. Chapin and W. C. Powick III

mentioned, and by the picric acid method, 200 cc. of the extract being used for the picric acid treatment, and 100 cc. for each of the other methods. The total volume of the solution in the picric acid method was determined, after expelling the thymol, by Modification C. The following results were obtained:

PER CENT OF 80LUBLE INORGANIC P201 fN

METHOD OBIGINAL ME.4T

Results of separate determinations Average

Forbes magnesia mixture.. . . . . . . 0.2856 0.2848 0.2838 0.2848 Emmett-Grindley.. . . . . . . 0.2792 0.3224* 0.2794 0.2793 Siegfried-Singewald.. . . . . . . 0.2812 0.2800 0.2806 0.2806 Picric acid.. . . . . . . . . 0.2750 0.2746 0.2740 0.2745

* Rejected.

Esperiment B. The previous experiment was repeated, except that the meat used for the preparation of the extract was somewhat older, and that Modification B instead of Modification C was used for the picric acid method. In using Modification ,B the pi&c acid was added after the solution had been brought to definite volume, and no correction for volume was then found to be necessary. The results obtained in this experiment are given below: -

METHOD

Forbes magnesia mixture. ......... Emmett-Grindley. ................. Siegfried-Singewald. ............... Picric acid .........................

Results of separate determinationa Average

0.3519 0.3533 0.3523 0.3525 0.3531 0.3536 0.3546 0.3538 0.3506 0.3522 0.3487 0.3505 0.3463 0.3459 0.3471 0.3464

In each of the above experiments lower results were obtained by the picric acid method than by any of the other methods used. It is believed, however, that the results of the determinations by our method are quantitative, since the method is theoretically correct and has been indicated to be accurate by the experiments already recorded and by numerous others which we have not brought forward. The larger results obtained by the other methods are to be explained, we believe, by the precipitation of some of the organically combined phosphorus, or by the liberation of inorganic phosphorus from organic phosphorus compounds, or both.


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II2 Estimation of Inorganic Phosphoric Acid

The distribution of phosphorus in eggs.

A few hitherto unpublished results obtained by the use of the picric acid method on eggs are here appended. It was our object in these experiments to determine the ratio between the inorganic and the total phosphorus in different grades of eggs, in order to see if there might exist any relation between this ratio and the degree of deterioration as judged by candling. The experiments showed a progressive increase in this ratio as deterioration advanced. The method used for the determination of the inorganic phosphoric acid was the above proposed picric acid method, Modification A being employed. The total phosphoric acid was determined by igniting the dried egg substance with soda and saltpeter, and, after dissolving the residue and filtering, by precipitating by the Lorenz method. The results obtained are shown in Table 7.

CONCLUSION.

1. An improved method for the determination of inorganic phosphoric acid in tissues has been devised, which, on a priori grounds, should yield correct results, and which has the following advantages over the methods customarily used:

a. It does not. involve the action of heat or of strong reagents upon the organic matter present in the tissue.

b. Bacterial decomposition of the organid matter is prevented by the presence of picric acid, a powerful antiseptic.

c. The precipitation is effected after the removal of the proteid matter, and therefore is not influenced by the protective action of colloids; while contamination with organic compounds of phosphorus is reduced to a minimum.

d. Tedious filtrations are avoided by using an aliquot portion of the filtrate from the picric acid coagulum for the determination of inorganic phosphoric acid.

e. A rapid and accurate chemical method has been employed for determining the proportional part of the whole solution represented by the aliquot part used.

2. The method described, and its several modifications, have been experimentally tested and found to be rapid and accurate.


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TABLE 7.

Distribution of vhosvhorus in di.fferenl grades of eggs. ,-

Fresh eggs, 24 hrs. old

73.91

0.0165 0.0633 0.0159 0.0610 0.0159 0.0612 --___~ 0.0161 0.0618

Fresh eggs, 24 hrs. old

- 73.91

Fresh eggs, 24 brs. old

“Fresh” eggs, aa judged by candling test

Second grade eggs, kept frozen for 185 dye.

- - E I *OF I g I 2: - -- 49

- -- 50

- -- 55

-- -

52

- -- 57

- _- (

58 (

I _-

59

54 , - 7

-

23 E - a

74.3: 74.3: - 74.34 - 73.71 73.6; -

73.66 -

72.72 72.67

0.0179 0.0689 0.0179 0.0686 0.0183 0.0794 0.0182 0.0703 0.0183 0.0704 0.494 1.900 ~-~-~ 0.0181 0.0697 0.494 1.900 3.670 ----- 0.0179 0.0700 0.493 1.922 0.0180 0.0703 0.496 1.935 ___-__-__ 0.0180 0.0701 0.495 1.929 3.555 -----

0.510 1.939 0.0156 0.0592 0.507 1.928 ~________

3.0156 0.0592 0.509 1.933 3.064 _____ ______

I.0227 0.0830 0.543 1.988 I.0229 0.0838 0.546 2.002

1.0228 0.0834 0.545 1.995 4.181

Poorer grade of No. 2 eggs kept frozen for 187 dys.

j.0207 0.0687 0.490 1.607 j.0209 0.0679 0.492 1.615 ---~__ I.0208 0.0683 0.491 1.611 4.239

“Borderline,” almost inedible eggs, kept frozen for 187 dys.

Shell eggs, containing dead, 2 dy. old embryo; in incubator two wks.

72.7C - 39.67 39.38 -

39.52 -

74.02 73.52 -

73.77 -

10.67 ‘0.72 - ‘0.70 -

j.0279 0.1062 0.627 2.392 j.0279 0.1063 0.619 2.360 _______~~

1.0279 0.1063 0.623 2.376 4.437 ________-

) .0408 0.550 1.879 I.0418 0.553 1.886 ~__-~~~

1.0413 0.1409 0.552 1.882 7.486

“Rots,” egga in a well advanced stage of de- i8.93

-_

._ 3 .-

1 .- 7 !

L .- L f / .- , I

I ( ’ ( .-

I I -

( (

-

( - ( (

( -

( (

-

C -

(I 0 1.1294 0.4163 0.536 1.725 1.1280 0.4119 0.546 1.758

is.93 0.1287 0.4141 0.541 1.741 23.78 -1777-i:


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3. Experiments for the comparison of the picric acid method with the Forbes, the Emmett-Grindley, and the Siegfried-Singe- wald methods have shown that approximately the same results are obtained by each, 1 when employed for the determination of inorganic phosphorus in the cold water extract of flesh. The slightly lower results obtained by the picric acid method are attributed to the sharper separation of the inorganic from the organic phosphorus compounds that can be obtained by its use.

4. By rqeans of this method, a progressive increase in the ratio of the inorganic to the total phosphorus in eggs has been found, which increase corresponds to the increased deterioration of the eggs as judged by physical means.


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Robert M. Chapin and Wilmer C. PowickTISSUES AND FOOD PRODUCTSPHOSPHORIC ACID IN CERTAIN

ESTIMATION OF INORGANIC AN IMPROVED METHOD FOR THE

1915, 20:97-114.J. Biol. Chem.

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