LESSON XXV

ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE

ESTIMATION OF PHOSPHATES

The phosphoric acid in the urine is combined with soda, potash, lime, and magnesia.

(a) Estimation of the total phosphates.

For this purpose the following reagents are necessary:—

i. A standard solution of uranic nitrate. The uranic nitrate solution contains 35.5 grammes in a litre of water; 1 c.c. corresponds to 0.005 gramme of phosphoric acid (P2O.).

ii. Acid solution of sodium acetate. Dissolve 100 grammes of sodic acetate in 900 c.c. of water; add to this 100 c.c. of glacial acetic acid.

iii. Solution of potassium ferrocyanide.

Method.-Take 50 c.c. of urine. Add 5 c.c. of the acid solution of sodium Heat the mixture to 80° C.

acetate.

Run into it while hot the standard uranium nitrate solution from a burette until a drop of the mixture gives a distinct brown colour with a drop of potassium ferrocyanide placed on a porcelain slab. Read off the quantity of solution used and calculate therefrom the percentage amount of phosphoric acid in the urine.

(b) Estimation of the phosphoric acid combined with lime and magnesia (alkaline earths).

Take 200 c.c. urine. Render it alkaline with ammonia. Lay the mixture aside for twelve hours. Collect the precipitated earthy phosphates on a filter; wash with dilute ammonia (1 in 3). Wash the precipitate off the filter with water acidified by a few drops of acetic acid. Dissolve with the aid of heat, adding a little more acetic acid if necessary. Add 5 c.c. of the acid solution of sodium acetate. Bring the volume up to 50 c.c., and estimate the phosphates in this volumetrically by the standard uranium nitrate as before. Subtract the phosphoric acid combined with the alkaline earths thus obtained from the total quantity of phosphoric acid, and the difference is the amount of acid combined with the alkalis soda and potash.

(c) Instead of uranium nitrate a standard solution of uranium acetate may be used. The directions for the making of these standard solutions will be found in Sutton's 'Volumetric Analysis.' As a rule, it is less troublesome, and not much more expensive, to purchase standard solutions readymade.

ESTIMATION OF SULPHATES

The sulphates in the urine are of two kinds: the pre-formed sulphate viz. those of soda and potash, and the combined or ethereal sulphates.

ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE 145

(a) For the determination of the total amount of sulphuric acid (SO) (i.e. pre-formed and combined sulphuric acid together) in the urine, one of two methods is adopted :--

1. Volumetric method.

2. Gravimetric method.

Both methods will be given here; the former is, however, better suited for class experiments.

1. Volumetric Determination.--This process consists in adding to a given volume of the urine a standard solution of chloride of barium so long as a precipitate of barium sulphate is formed.

The following solutions are necessary :-

i. Standard barium chloride solution: 30.5 grammes of crystallised chloride of barium in a litre of distilled water; 1 c.c. of this solution corresponds to 0.01 gramme of sulphuric acid (SO).

ii. Solution of sulphate of potash: 20 per cent.

iii. Pure hydrochloric acid.

Method.-100 c.c. of urine are taken in a flask. This is rendered acid by 5 c.c. of hydrochloric acid, and boiled. The combined sulphates are thus converted into ordinary sulphates, and give a precipitate like them with barium chloride. The chloride of barium solution is allowed to drop into this mixture as long as any precipitate occurs, the mixture being heated before every addition of barium chloride to it. After adding 5 to 8 c.c. of the standard solution, allow the precipitate to settle; pipette off a few drops of the clear, supernatant fluid into a watch-glass; add to it a few drops of the standard barium nitrate solution. If any precipitate occurs, return the whole to the flask and add more barium chloride; again allow the precipitate to settle, and test as before; go on in this way until no more barium sulphate is formed on the addition of barium chloride.

Excess of barium chloride must also be avoided; when only a trace of excess is present a drop of the clear fluid removed from the precipitate gives a cloudiness with a drop of the potassium sulphate solution placed on a glass plate over a black ground. If more than a cloudiness appears, too large a quantity of barium chloride has been added, and the operation must be repeated. From the quantity of barium chloride solution used, the percentage of sulphuric acid in the urine is calculated.

2. Gravimetric Determination (i.e. by weight).—This method consists in weighing the precipitate of barium sulphate obtained by adding barium chloride to a known volume of urine; 100 parts of sulphate of barium correspond to 34.33 parts of sulphuric acid (SO).

Method (Salkowski).—100 c.c. of urine are taken in a beaker. This is boiled with 5 c.c. of hydrochloric acid as before.

Chloride of barium is added till no more precipitate occurs.

The precipitate is collected on a small filter of known ash, and washed with hot distilled water till no more barium chloride occurs in the filtratei.e. until the filtrate remains clear after the addition of a few drops of hydric sulphate. Then wash with hot alcohol, and afterwards with ether. Remove

L

the filter, and place it with its contents in a platinum crucible. Heat to redness. Cool over sulphuric acid in an exsiccator; weigh, and deduct the weight of the crucible and filter ash; the remainder is the weight of barium sulphate formed.

Error.-When the experiment is carried out as above there is a slight error from the formation of a small quantity of sulphide of barium. This may be corrected as follows: After the platinum crucible has become cool add a few drops of pure sulphuric acid (H2SO1). The sulphide is converted into sulphate. Heat again to redness to drive off excess of sulphuric acid.

(b) The following is Salkowski's' method of estimating the combined sulphuric acid--that is, the amount of SO, in ethereal sulphates:-100 c.c. of urine is mixed with 100 c.c. of alkaline barium chloride solution, which is a mixture of two volumes of solution of barium hydrate with one of barium chloride, both saturated in the cold. The mixture is stirred, and after a few minutes filtered; 100 c.c. of the filtrate (= 50 c.c. of urine) are acidified with 10 c.c. of hydrochloric acid, boiled, kept at 100° C. on the water-bath for an hour, and then allowed to stand till the precipitate has completely settled; if possible, it should be left in this way for twenty-four hours. The further treatment of this precipitate ( = combined sulphates) is then carried out as in the last case.

Calculation.-233 parts of barium sulphate correspond to 98 parts of H2SO4, or 80 parts or SO, of 32 parts of S. To calculate the H2SO4, multiply the weight of barium sulphate by = 0·4206; to calculate the SO, multiply

98 233

0.34335; to calculate the S multiply by = 0.13734. This method

32 233

of calculation applies to the gravimetric estimation both of total sulphates and of combined sulphates.

(c) To obtain the amount of pre-formed sulphuric acid subtract the amount of combined SO, from the total amount of SO. The difference is the preformed SO3.

Example: 100 c.c. of urine gave 0.5 gramme of total barium sulphate. This multiplied by = 0.171 gr. = total SO3. Another 100 c.c. of the same

80

233

urine gave 0.05 gr. of barium sulphate from ethereal sulphates; this mul

tiplied by =0·017 gr. of combined SO3. Total SO-combined SO,

80 233

Zeit. physiol. Chem. x. p. 346. This method is a modification of Baumann's original method, ibid. i. p. 71.

LESSON XXVI

URINE. MISCELLANEOUS EXERCISES

1. Estimation of Uric Acid.-Saturate a known volume, say 100 c.c., of urine with crystals of ammonium chloride. Collect the precipitate on a filter, wash it with saturated solution of ammonium chloride, and then dissolve the precipitate in weak alkali (2 per cent. potash).

Neutralise the alkali by the addition of hydrochloric acid. Uric acid is thus precipitated, and is collected on a weighed filter, dried, and weighed. The above method is that introduced by Hopkins. (For other methods see 'Chemical Physiology and Pathology,' pp. 807–809.)

2. Estimation of Creatinine. The crystalline compound which creatinine forms with zinc chloride is employed in estimating the quantity of creatinine in urine; 100 parts of the compound correspond to 62:42 of creatinine.

Method.-Take 250 c.c. of urine. Add milk of lime and calcic chloride in excess to precipitate the phosphates. Filter, and evaporate the filtrate to a small bulk; to this add 50 c.c. absolute alcohol, and let the mixture stand for six hours. Then add 10 or 15 drops of an alcoholic solution of zinc chloride; the crystals form, and after two or three days' standing in a dark place may be collected on a weighed filter.

Wash with 90 per cent. alcohol, dry and weigh, and thence calculate the percentage of creatinine. (For CREATINE and CREATININE see 'Chemical Physiology and Pathology,' pp. 84, 418, 421, 726, 727, 737, 813.)

3. Urobilin.-Add neutral and then basic lead acetate to urine until there is no further precipitate. This precipitate carries down most of the pigment with it. Filter; the filtrate is clear and almost colourless. Extract the urobilin from the precipitate by alcohol acidulated with sulphuric acid; filter. The filtrate is yellow. Agitate it with chloroform and examine the chloroformic solution (concentrated, if necessary) with the spectroscope. Note the shading in the region of the F line (see Chemical Physiology and Pathology,' Chap. XLI. p. 747).

4. Preparation of Urea from Urine.-1. Evaporate the urine to a small bulk. Add strong pure nitric acid in excess, keeping the mixture cool during the addition of the acid. Pour off the excess of fluid from the crystals of urea nitrate which are formed; strain through muslin and press between filter paper. Add to the dry product barium carbonate in large excess. This forms barium nitrate and sets the urea free. Mix thoroughly with sufficient methylated spirit to form a paste. Dry on a water-bath and extract with

alcohol; filter; evaporate the filtrate on the water-bath and set aside. The urea crystallises out, and may be decolourised by animal charcoal and purified by recrystallisation.

2. The following method is well adapted for the preparation of microscopic specimens of urea and urea nitrate: Take 20 c.c. of urine; add 'baryta mixture' (two volumes of barium hydrate solution and one volume of barium nitrate solution, both saturated in the cold) until no further precipitate is produced; filter, evaporate the filtrate to a thick syrup on the water-bath, and extract with alcohol; pour off and filter the alcoholic extract; evaporate it to dryness on the water-bath and take up the residue with water. Place a drop of the aqueous solution on a slide and allow it to crystallise; crystals of urea separate out. Place another drop on another slide and add a drop of nitric acid; crystals of urea nitrate separate out.

ment.

5. Preparation of Pure Uric Acid.-If one wishes to prepare pure uric acid, the solid urine of a reptile or bird, which consists principally of the acid ammonium salt, should be selected; one has not then to separate any pig. It is boiled with 10 per cent. caustic soda or ammonia, diluted, and then allowed to stand. The clear fluid is decanted and poured into a large excess of water to which 10 per cent. of hydrochloric acid has been added; after twenty-four hours, crystals of uric acid are deposited. These may be purified by washing, re-solution in soda, and re-precipitation by acid.