ARTICLE: MICRO COLORIMETRIC STUDIES: I. A MOLYBDIC ACID, STANNOUS CHLORIDE REAGENT.

THE MICRO ESTIMATION OF PHOSPHATE AND CALCIUM IN PUS, PLASMA, AND SPINAL FLUID Theodore Kuttner and Harriet R. Cohen
J. Biol. Chem. 1927, 75:517-531.

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MICRO

COLORIMETRIC

STUDIES.
THE IN

I. A MOLYBDIC ACID, STANNOUS CHLORIDE REAGENT. MICRO ESTIMATION OF PHOSPHATE AND CALCIUM PUS, PLASMA, AND SPINAL FLUID. BY THEODORE KUTTNER*
AND

HARRIET

R. COHEN.

(From the Laboratories

of the Mount Sinai Hospital, New York.)

July 21, 1927.)
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(Received for publication,

During the course of certain biological and pharmacological investigations in this laboratory, it was desirable to have at hand a method for the estimation of small amounts of phosphorus, arsenic, and antimony. As is well known, when these substances in the pentavalent state are acted upon by a reducing agent in an acid mixture in the presence of molybdic acid, a blue solution results. The intensity of color varies proportionately to the amount of phosphorus, arsenic, or antimony present. This reaction has been made the basis of numerous quantitative methods for their estimation, but it is believed that until now the optimum conditions have not been established. It may be stated at the outset that we were looking for a stable substance which would act as reducing agent and produce the maximum intensity and stability of color in the cold without undue loss of time.
A calorimetric method for phosphorus in which molybdic oxide is used, was published by F. Osmond (1) in 1337, in which the washed, precipitated ammonium phosphomolybdate is reduced with stannous chloride. Taylor and Miller (2), in 1914, used phenylhydrazine to reduce the ammonium phosphomolybdate precipitated from an ashed sample. Hydroquinone was employed by Bell and Doisy (3) in their method for estimating phosphorus in blood. The reaction Icould be carried out with an excess of molybdate ions and it was found unnecessary to isolate the phosphate as ammonium phosphomolybdate. Briggs (4) modified the method by addition of a little sodium sulfite to the reagent and carried out the reaction in an acid medium, thus obtaining a better blue coloration with greater stability. Deniges (5), at about the same time as Briggs, used 4 drops of an ammo* Eugene Meyer, Jr., Fellow. 517

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Calorimetric

Studies.

nium molybdate-sulfuric acid mixture and 2 drops of a freshly prepared stannous chloride solution, and wafited 10 minutes before comparing colors. Stanford and Wheatley (6) have t#ested Briggs method and found it to be accurate and the reaction to be trustworthy for quantitative estimation of phosphorus, maximum intensity of color being reachedin 30 minutes. Benedict and Theis (7) have introduced a method, whereby boiling the mixture of molybdic and sulfuric acidswith the phosphate completes the reaction in 15 minutes. They retained the hydroquinone and sulfite reducing agent. Fiske and Subbarow (8) have recommended aminonaphtholsulfonic acid instead of hydroquinone. The 1,2,4- and the 1,4,6acids are equally effective. They used sulfite as well as bisulfite of soda and obtained maximum intensity in 5 minutes in the cold.

Where sulfuric acid is substituted for the sulfite or bisulfite to prevent oxidation of the hydroquinone, as has been done occasionally, it is less effective and the solution becomes brown in a few days. In the present investigation many substances were tested which act as reducing agents. Among them were hydroquinone, diaminophenol, p-aminophenol, monomethyl-paminophenol, monochlorohydroquinone, p-aminosaligenin (edinol), 1, 2, 4- and 1, 6, 8- aminonaphtholsulfonic acids, and stannous chloride. The last is by far the best when the concentrations of the reagents used are properly regulated. The effect of varying concentrations was studied as well as certain conditions interfering with the maximum development of the color. 1. Molybdic-Sulfuric Acid and Stannous Chloride Reagents.

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A. Use of Sulfuric Acid in Method.-In agreement with Stanford and Wheatley it was found that in the Briggs method, gradually increasing concentrations of sulfuric acid caused an increase in color until a maximum was reached, then with greater concentrations of the acid the color intensity decreased. In the present method the intensity as well as the purity of the color is regulated by varying the concentration of the reagents. The optimum acidity of sulfuric acid as shown by Fig. 1 for reduction of the phosphomolybdic acid lies in a zone between 0.9 and Below this optimum acidity, molybdic 1.05 N in the final mixture. acid is itself reduced; the lower the concentration of sulfuric acid, the greater the reduction of molybdic acid. An increase of acidity above the optimum causesat first a retardation in the development of the color, then with further increase an inhibition of color production occurs. Thus at 1.1 N acidity, it requires about 2 minutes

T. Kuttner

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519

N HzSO, X Per cent coloration. 100 75 50

I
0.9

0.8

0.7

.w-

35 FIQ. 1. Effect of increasing concentration of sulfuric acid. Zone la = decreased color production of 35, 50, and 75 per cent, also increase of turZone lb = delayed maximal color producbidity with increase of acidity. tion, of 5 minutes at 5, to 2 minutes at 2. Zone 2 = maximal color production in 15 seconds. Zone 3 = production of additional color by reduction of Moos.
#

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Per cent sodium molybdate. 0.63 0.65 Per cent coloration.

Zone 1 0.70

I 0.73

I 0.75

3 0.80

100 80 75 50 350"

FIG+. 2. Effect of increasing concentration of sodium molybdate. Zone 1 = decreased coloration. Zone 2 = optimum for maximal color. Zone 3 = zone of additional color by reduction of MOOS. Zone 1 0.01 0.02 0.022

Per cent stannous chloride. 0.005 Per cent coloration. 1: *-+

Fro. 3. Effect of increasing concentration of stannous chloride. 1 = decreased coloration. Zone 2 = optimum for maximal color. 3 = additional color by reduction of Moos.

Zone Zone

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Calorimetric

Studies.

to reach maximum intensity, at 1.2 N about 5 minutes, while at 1.3 and 1.4 N the intensity of coloration is lessened to 75 and 50 per cent respectively. In addition to this retarding effect an increasing turbidity develops as the acidity is increased beyond 1.2 N. The acidity of the final mixture in our method is about 1 N sulfuric acid; that in the Briggs method is 0.27 N, in the Fiske and Subbarow procedure 0.5 N, in the Benedict and Theis method about 1.9 N, and in that of Roe and Kahn (9) about 1.4 N. B. Effect of Increasing Molybclic Acid Concentration.-A 7.5 per cent solution of sodium molybdate was found convenient for this study. The optimum concentration of sodium molybdate in the final mixture (as shown in Fig. 2) lies in a zone between 0.73 and 0.75 per cent of sodium molybdate. Less than this amount effects a decrease in color production, while above this concentration the molybdic acid is reduced in direct proportion to the increased amount. C. Stannous Chloride.-Stannous chloride, though used as a reducing reagent in qualitative calorimetric analysis, has not found favor in quantitative work. This is perhaps because yellowish brown molybdic oxide complexes are produced simultaneously with the blue color, but in varying amounts, thus resulting in olivegreen tints. This disturbing influence has been eliminated and the use of sulfite found unnecessary. The optimal concentration of stannous chloride lies in a zone between 0.02 and 0.022 per cent, as shown in Fig. 3. Stronger solutions reduce molybdic oxide as well as phosphomolybdic acid.
2. Interfering Substances.

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Interfering substances can retard color production, inhibit it partly or entirely, or develop a different color in addition. The degree of interference depends upon the quantity of the disturbing substance. For example, trichloroacetic acid begins to interfere with maximum color production only at concentrations above 4 per cent in the final mixture. Hydrochloric acid has a tendency to lessen color stability and retards and inhibits maximum coloration. Thus 2 N hydrochloric acid in the final mixture inhibits color production. N hydrochloric acid retards and prevents maximum coloration, while 0.5 N hydrochloric acid allows maximum produc-

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521

tion of the color but it soon fades. Nitric acid interferes above 0.003 per cent, and with a large amount, for instance 1.5 per cent, a green coloration results. Tartaric acid interferes above 0.002 per cent with inhibition of maximum color, and above 0.04 per cent with reduction of molybdic acid itself. The effect of citric acid is twice as great. Hypochlorites interfere strongly. Up to 0.00004 per cent there is no difference in intensity but a slight difference in the tint, while at 0.00008 per cent a 20 per cent loss of color occurs, and there is a 50 per cent loss of color in the pres?ence of 0.0004 per cent. Nitrites interfere strongly at concentrtG tions greater than 0.0001 per cent. Traces of copper or iron salts do not interfere. Sulfates interfere presumably by depressing the ionization of the sulfuric acid, the interference depending upon the quantity present. Salts of weak acids such as acetates, tartrates, and citrates also interfere, whereas acetic acid itself does not interfere, but causesthe development of purplish tints at concentrations above 5 per cent. Silicates do not interfere at the optimum acidity, up to 4 or 5 times the amount of phosphorus present.
3. General Considerations.

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The test is sufficiently sensitive to determine about 0.001 mg. of phosphorus, arsenic, or antimony in the pentavalent state in 5 cc. of solution. To prevent misleading results, with so sensitive a method, it is obviously necessary to work with pure reagents. The blue color is produced practically instantaneously, without heating, the reaction being complete in 15 seconds when conditions are right. The velocity of the reaction is about 5 to 10 times greater than in the Fiske and Subbarow method, about 40 times that of the Deniges procedure, and more than 100 times that of the Briggs or Benedict and Theis methods. The colors produced by the other methods mentioned have a purplish tint, while the present method yields a better blue with greater intensity. There is no parallelism between the intensities and velocities of the colors produced by the different methods. The estimation of the comparative value of the colors is somewhat difficult be:ause of the difference in tint. In the new method the color is approximately 3 to 4 times greater than that of the Benedict and Theis or

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Calorimetric

Studies.

Deniges methods, and about 12 to 15 times that of the Briggs or the Fiske and Subbarow methods. Based upon the reaction mentioned in this paper, methods for the quantitative determination of phosphorus, arsenic, antimony, and calcium, etc., have been devised. Only methods for phosphates and calcium will be considered in this paper.

4. Description

of Methods.

A. Method for Estimation of Phosphates.-The following solutions are necessary and thus far they have remained unchanged after severalmonths. 1. Molybdic-Sulfuric Acid Mixture.-Dissolve 18.75 gm. of sodium molybdate in 2.5 N sulfuric acid; or mix 1 volume of 7.5 per cent sodium molybdate (Kahlbaums Zur Analyse) with 1 volume of 10 N sulfuric acid, and add 2 volumes of distilled water. Store in a brown, glass-stoppered bottle. 2. Stannous Chloride Stock Solution.-Dissolve 10 gm. in 25 cc. of concentrated hydrochloric acid. Store in a brown, glass-stoppered bottle. Dilute 0.5 cc. of this stock solution to 100 cc. with distilled water. The diluted reagent does not keep, and should be freshly prepared as needed. 3. Standard Phosphate Stock Solution.-Dissolve 0.4394 gm. of dried monopotassium phosphate in 1 liter of distilled water. Add a few drops of chloroform to prevent mold formation. 1 cc. = 0.1 mg. of phosphorus. Make two standard phosphate solutions by diluting 5 cc. and 10 cc. of the phosphate stock solution in 100 cc. graduated flasks and fill to the mark with water. The solutions will contain respectively 0.005 and 0.01 mg. of phosphorus per cc. The solution to be tested should contain between 0.4 and 1.2 mg. of phosphorus per 100 cc. (1) Place 2.5 cc. of the sample to be examined in a test-tube graduated at 5 and 10 cc. The sample should contain between 0.01 and 0.03 mg. of phosphorus. (2) Place2.5 cc. of each standard phosphate solution in two other similarly graduated test-tubes, so that one tube contains 0.0125 mg. and the other 0.025 mg. of phosphorus. (3) Add to each tube 2 cc. of the molybdie-sulfuric acid reagent and mix. (4) Add simultaneously to each of the three tubes 0.5 cc. of the diluted stannous chloride reagent. Insert rubber stoppers and mix without delay by inverting the tubes once or twice.

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The color is produced immediately, and although the reaction is complete in 15 seconds if conditions are right, it is advisable to wait a full minute before comparison with the standards is made. Any type calorimeter can be used but it may be necessary to dilute The method all solutions to 10 cc. if a plunger type is employed. of procedure and calculation is carried out in the manner usual for the type of calorimeter employed. B. Method for Estimation of Calcium.-The estimation of calcium is similar to that of Roe and Kahn (9), the calcium being precipitated as phosphate and the phosphorus estimated colorimetrically. It differs however from theirs by the omission of phenolphthalein and by the use of a standardized alkaline phosphate solution for the precipitation. A phosphate standard expressed in terms of calcium is also used in the present method. Whether the concentration of alkali present for precipitation has an influence on the character of the precipitate is being investigated, as well as the solubility of calcium phosphate. It appears that under certain conditions the precipitate does not consist of tertiary calcium phosphate Caa(PO& only, but of a mixture containing also dicalcium phosphate Ca2H,(PO&. The mixture may The ratio of calcium to vary in the proportion of each substance. phosphorus in the tricalcic phosphate is approximately 4:2, in the dicalcic phosphate 4 : 3. The latter contains 25 per cent more phosphorus with subsequent greater color production. It is obvious that a precipitate containing a mixture of the two would give erroneous results when compared with a standard prepared with pure calcium salt under different conditions. The most favorable amount of calcium present for the test to be described is a concentration of about 0.4 to 1.2 mg. per 100 cc. of solution. The following solutions are necessary: I. Molybclate-Sulfuric Acid Mixture. 2. Diluted Stannous Chloride Reagent.-Reagents 1 and 2 have been described above under the phosphate method. 3. 10 Per Cent Solution of Sodium Hydroxide Containing 1 Per Cent of Basic Trisodium Phosphate.-Dissolve 1 gm. of the phosphate in 50 cc. of distilled water and mix with 50 cc. of 20 per cent If a precipitate sodium hydroxide (free from silica and calcium). forms it should be allowed to settle for 24 hours or a small portion

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Micro

Calorimetric

Studies.

may be centrifuged in an hour for immediate use. In the Roe and Kahn method the phosphate and hydroxide solutions are used separately and may therefore give rise to error in estimating the true calcium value. Q. 50 Per Cent Solution of Alcohol.-Dilute 55 cc. of 95 per cent alcohol with water. Make faintly alkaline with calcium-free sodium hydroxide, litmus paper being used as indicator. Make up to 100 cc. 5. Two Standard Phosphate Solutions.-(A) Dilute 51.7 cc. of the phosphate stock solution described in the phosphate method to 1000 cc. in a graduated flask. 2.5 cc. are equivalent to 0.025 mg. of calcium. (B) Dilute 51.7 cc. of the phosphate stock solution in a graduated flask to 500 cc. 2.5 cc. are equivalent to 0.05 mg. of calcium. Transfer 5 cc. of the solution of the sample to be analyzed to a centrifuge tube or Pyrex test-tube graduated at 5 and 10 cc. The solution should have a calcium content of about 0.02 to 0.06 mg. and an acidity of not more than 7 per cent trichloroacetic acid, as previously described. Add 1 cc. of the alkaline sodium phosphate mixture, mix, set aside for 1 hour, then centrifuge for 3 minutes. Discard the supernatant fluid, catching the last drop on a piece of blotting paper. Allow to drain 1 to 2 minutes, then wipe the rim of the tube, and wash twice with 5 cc. portions of the faintly alkalinized alcohol, being careful in so doing also to rinse the sides Drain of the tube and to break up the phosphate precipitate. Add 2 cc. of and wipe the tube after each centrifuging as before. the molybdic-sulfuric acid mixture to the tube and dissolve the Add 2.5 cc. of water and mix. If desired 4.5 cc. of precipitate. a diluted molybdate mixture can be used, prepared by diluting 200 cc. of the molybdate-sulfuric acid mixture with 250 cc. of distilled water. Into one of two graduated test-tubes transfer 2.5 cc. of calcium Standard A and 2.5 cc. of Standard B into the other; add 2 cc. of molybdic-sulfuric acid mixture to each, and mix. Add 0.5 cc. of the diluted stannous chloride reagent to each standard as well as the unknown and without delay close with a rubber After about 1 minute stopper and invert each tube once or twice. If the plunger type of calorimeter is compare in a calorimeter. used proceed in the same manner as described for phosphorus. Described below is an adaptation of the method on a small

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525

scale, devised for the estimation of phosphates and calcium in 0.1 to 0.2 cc. of blood plasma, spinal fluid, or pus. Calcium may even be determined if only 0.05 cc. of pus is available. The micro method was primarily devised for calcium determinationson the usually small amounts of pus obtained from the human ear in diseased conditions such as mastoiditis. Friesner and Rosen (10) have shown that pus derived from bone destruction is relatively high in calcium, and their investigation was the stimulus for the development and use of the present micro method. Pus, unlike plasma, is not a homogeneous fluid and may contain debris from bone or soft tissue. The calcium cannot be directly isolated as oxalate, as Kramer and Tisdall (11) do in the case of blood plasma, even if a sufficient quantity is available for the use of their method. In addition, the calcium isolated as oxalate from the ash of a small sample is too minute in amount to be titrated with potassium permanganate. Less material is required with the Van Slyke and Sendroy (12) gasometric method, in which 1 cc. of blood plasma is sufficient and even less may be taken. Roe and Kahn (9) use 2 cc. of plasma and estimate the phosphorus by the Benedict and Theis (7) method from the precipitated tertiary calcium phosphate. This procedure was tested on ashed samples of small quantities of material. In the use of this method, the exact matching of color depth with the Dubosq calorimeter was prevented because of the somewhat variable greenish tints in the unknowns. The influence of the adventitious yellow is increased and becomes more disturbing as the depth of the liquid increases. This effect is lessened and minimized by the use of a dilution type of calorimeter such as the micro calorimeter (13) described in a previous paper. This instrument also permits employment of smaller samples and less reagent for the test. Ultimately it was found that phenolphthalein is the disturbing factor, being adsorbed by the calcium precipitate, and the indicator is therefore omitted. Although the greenish tint is entirely eliminated in the new method, the micro calorimeter is retained because of convenience, equal accuracy, and other advantages. The old type of diluting tubes has been replaced by glass-stoppered tubes1 of uniform bore, graduated to 180 on an etched
1 The colo&eter and glassware are manufactured East 10th Street, New York. by E. Leitz, Inc., 60

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Micro

Calorimetric

Studies.

scale, each division on the scale equalling 0.02 cc. The use of permanent color standards has been found very convenient, and their preparation will be described in another paper. A pipette furnished with a rubber nipple; and drawn to a fine bore at the lower end, replaces the old type, permitting the use of smaller A narrow test-tube drops for dilution or a fine stream for rinsing. (12 X 120 mm.) graduated at 1 and 2 cc. has been found useful for the precipitation of the protein. Micro Estimation of Phosphates.

Two permanent standards are used: A, equivalent to 0.0025 mg. of phosphorus, and B, equivalent to 0.005 mg.; or if desired, color standards may be prepared from the standard phosphate solutions, but corresponding to these amounts. Procedure. (1) Transfer 0.2 cc. of pus or blood plasma to the narrow testtube already described. (2) Add 7 per cent trichloroacetic acid to the 2 cc. maPk,2 close with a small rubber stopper, shake, and centrifuge after a few minutes. (3) Transfer 0.5 cc. of, the clear &d colorless fluid, equivalent to 0.05 cc. of the original sample, to the glass-stoppered, graduated, diluting tube of the micro colorimeter. (4) Add 0.4 cc. of the molybdic-sulfuric acid solution and (5) Add 0.1 cc. mix by sharply tapping the lower end of the tube. of the diluted stannous chloride reagent, close with the glass stopper, and invert the tube at once. The color is produced immediately, full intensity being reached After 1 minute, compare in 15 seconds under optimum conditions. with the permanent color standards or with freshly prepared standards run through in the same manner simultaneously with the unknown. The unknown is compared with the proper standards in the micro calorimeter by cautious dilution with small quantities of water with the aid of the special pipette. Any other calorimeter may be used if the unknown and standards are diluted to a suitable volume. The method of computation using dark Standard B in the micro calorimeter is simple. Each division on the scale represents 0.1
* If 0.1 cc. of material has been taken make up to the 1 cc. mark.

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T. Kuttner

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mg. of phosphorus in 100 cc. of the original material taken for analysis. For example, if the colors match exactly with the meniscus in the diluting tube at 56, then 56~ 10 equals 5.6 mg. of phosphorus in 100 cc. of the material taken. If the color had been matched with the light standard each division would represent 0.05 mg. per 100 cc. and the reading would be divided by 20, thus 56+20 is 2.8 mg. When working with material relatively low in phosphorus and protein such as spinal fluid, for example, 0.2, 0.25, or 0.5 cc. is made up to 1 cc. only with the trichloroacetic acid. The reading is now divided by 40,50, or 100 as the case may be. F = amount of original sample actually used for calorimetric S = amount of P or Ca in standard. R = reading of unknown on graduated scale.
ThenPorCaper 100~. =F comparison.
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For example, if F = 0.05 cc., R = 56, and S = 0.005 mg. of P or Ca, then in 100 cc. original sample
PorCa=Oq5X56X$

X 0.005= 5.6 mg. per 100 cc.

With the use of permanent standards .a correction should be made by adding 1 per cent for every degree above 21C or subtracting below 21C. The smallest amount of phosphorus that can be determined in this manner is 0.25 mg. in 100 cc. Micro Estimation of Calcium. Two permanent standards are used : A, equivalent to 0.01 mg. of calcium, and B, equivalent to 0.005 mg. If desired, color standards may be prepared for calcium from the. standard phosphate solutions, but corresponding to these amounts. An accurately calibrated 0.2 cc. pipette is used, graduated in hundredths and of a bore sufficiently large for thick pus should such be encountered. Procedure. Transfer 0.1 cc. of pus or blood plasma with the above pipette to a small platinum dish, rinse the pipette several times with water,
* 1 unit of the graduated scale.

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Micro

Calorimetric

Studies.

and add the rinsings to the dish. Evaporate to dryness, cautiously burn off all organic matter, add a drop or two of concentrated nitric Care must be taken of acid, and again ignite in the usual manner. course to prevent spattering during the process, which can be comAfter cooling, dissolve the precipitate pleted in about 10 minutes. with 7 per cent trichloroacetic acid in successive small portions of about 0.2 to 0.3 cc. each. The dish is rinsed down the sides with each portion which is then transferred quantitatively to the narrow test-tube, with the aid of the special pipette drawn to a fine bore at the lower end. The total volume should not be much over 1 cc. The calcium may be precipitated from this solution or from 1 cc. of the deproteinized filtrate as obtained in the micro method for
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TABLE I.

Mg. of calcium per 100 cc.

Ashed. -~ Triohloroacetic acid.

I bhosphoric _-

Irichloroacetic + acid.

Bloodserum............................... . .. ... ... ... .. ... ... ... ... ... .. . .. ... ... ... .. ... ... ... ... ... .. I . .. ... ... ... .. ... ... ... ... ... .. Pus, empyema, chest. . . . ... . . . . . . . .,. . . . . . . . I I 8 mo. later. . . . . . . . . . ic osteomyelitis.. . . . . . . . . . . . . . . . . . . . . . . . . I I . . . . . . . . . . . . . . . . . . . . . ... . .

11.5 10.0 10.0 12.5 45.6 14.6 19.8 13.5

9.5 8.0 8.5 10.4 32.0 13.8 17.4 12.2

-

8.8 11.0 38.0 14.2 18.9 13.1

phosphate determination. In the latter casethe results are somewhat lower, but are generally within 10 per cent of those obtained by the ashing method. When 0.5 cc. of syrupy phosphoric acid is added to the liter of 7 per cent trichloroacetic acid, the result is better. This is shown in Table I. In either caseproceed as follows: (1) Add 0.2 cc. of the alkaline sodium phosphate mixture and set aside for 1 hour. Centrifuge 3 minutes at high speed and discard the supernatant fluid, catching the last drop on a piece of blotting paper. Allow to drain 1 to 2 minutes and wipe the rim of the tube. Wash twice with 1 cc. portions of the faintly alkalinized alcohol, rinsing the sidesof the tube free from phosphates. Drain and wipe the rim of the tube after each centrifuging as before. When no colored indicator is used in

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the reagents it is difficult to see the slight precipitate, but this need not cause concern. (2) Add 0.4 cc. of the molybdic-sulfuric acid reagent and dissolve the precipitate, then add 0.5 cc. of distilled water. If desired, 0.9 cc. of a diluted reagent may be used in the proportion of 40 cc. of the molybdic-sulfuric acid to 50 cc. of distilled water. (3) Add 0.1 cc. of the diluted stannous chloride reagent, close the tube with a rubber stopper, and invert at once. After 1 minute, transfer the solution with the aid of the special pipette to the graduTABLE II.

Mg. of calcium per 100 CC.

KEY Tisdall.
Solution of CaCl~, Containing 10 mg. Ca per 100 cc. ........................... Solution of CaCle, containing 15.1 mg. Ca per 100 cc. ........................ 1 Blood serum (ashed). ...................... ............................... ............................... L + 10 mg. as CaCla per 100 cc .... ............................... ............................... tetany. ... :. ................. ........................ I ............................... Pus, empyema, chest. ..................... . ....................... ............................ Spinal,fluid, infection ...................... 10.2 9.9 15.2 14.8 11.4 10.4 20.2 12.0 15.0 15.1 11.5 9.6 10.5 9.0 5.0 6.7 11.0

Microcolorimetric.

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11.5 6.4 6.0 45.2 6.0

10.0 10.2 15.2 15.0 11.5 9.5 10.5 20.4 12.0 9.2 5.1 6.8 11.3 6.3. 7.0 45.6 6.5

ated diluting tube of the calorimeter, and compare with the standards. The colors are matched in the same manner as described for phosphorus. The computation is exactly similar, except that the final result must be multiplied by 2 if only 0.05 cc. of material has been ashed. As shown in Table II the method is accurate and yields results comparable with those .of the Kramer and Tisdall (11) and the Van Slyke and Sendroy (12) methods.

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Micro

Colorirnetric
SUMMARY.

Studies.

A calorimetric method has been described for the estimation of phosphorus and calcium, based on the selective reduction of phosphomolybdic acid by stannous chloride when definite concentrations of reagents are maintained. Quantitative micro methods have been described for the estimation of phosphates and calcium in 0.1 to 6.2 cc. of material. As little as 0.05 cc. of pus may be used if only this amount is available. Newly designed glassware allowing greater accuracy has been added to the micro calorimeter (13). .A stannous chloride reagent is described~ which is more stable than others heretofore used. Amounts of phosphorus and calcium can be estimated ranging from 0.25 mg. per 100 cc. to 36 mg. and upwards, each individual test representing about 0.00125 to 0.009 mg. The advantages are greater rapidity in development of color, and a better blue and more uu%nse coloratioti. The method is more sensitive and permits the detection and determination of a smaller amount of phosphorus. and the use of less material than other methods heretofore published.
BIBLIOGRAPHY.

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1. Osmond, F., Bull. Sot. dim., 1887, x.X, 745. 2. Taylor, A. E., and Miller, C. W., On the estimation of phosphorus in biological material, J. Biol. Chem., 1914, xviii, 215. 3. Bell, R. D., and Doisy, E. A., Rapid calorimetric methods for the ,determination of phosphorus in urine and blood, J. Biol. Chem., 1920, xliv, 55. 4. Briggs, A. P., A modification of the Bell-Doisy phosphate method, J: Bibl.. Chem., 1922, liii, 13. 5. Deniges, G., Corn@. rend. Xoc. biol., 1921, lxxxiv, 875. 6. Stanford, R. V., and Wheatley, A. H. M., The estimation of phosphorus compounds in blood, Biochem. J., 1925, xix, 697. 7. Benedict, S. R., and Theis, R. C., A modification of the molybdic method for the determination of inorganic phosphorus in serum, J. Biol. Chem., 1924, lxi, 63. 8. Fiske, C.~ H;, and Subbarow, Y., The calorimetric determination of phosphorus, J. Biol. Chem., 1925, lxvi, 375. 9. Roe, J. H., and Kahn, B. S., A calorimetric method for the estimation of blood calcium, J. Biol. Chem., 1926, lxvii, 585.

T. Kuttner

and H. R. Cohen

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