Anal-yst, February, 1967, Vol. 92, $$. 107-111 107 The Determination of Phosphate and Calcium in Feeding Stuffs BY C. B. STUFFINS ( J . Bibby & Sons Ltd., Nutrition Research and Advisory Departments, “ Weatherstones,” Neston, Wirral, Cheshire) Procedures are described for the determination of phosphate and calcium in the Kjeldahl-digestion solution. PULSS~ y 2 described a procedure for the determination of phosphate in the Kjeldahl-digestion solution. A similar technique is adopted for the determination of phosphate, and also a procedure for the determination of calcium in the same digestion solution. The usual procedure adopted for the determination of protein, phosphate and calcium involves three main preliminary stages, vix., the weighing of at least two portions of the sample, i.e., one for protein and the other for phosphate and calcium determination; Kjeldahl digestion ; and dry-ashing.The following procedure eliminates one weighing in addition to a considerable amount of manipulative work connected with the dry-ashing technique, and also the use of crucibles and muffles. The elimination of much manipulative work will be appreciated in laboratories in which many of these analyses are carried out and to which the cost of an AutoAnalyzer would not be economically justified. To carry out the investigation, a feed mixture containing no added minerals was obtained, and known amounts of dicalcium orthophosphate were added to it. Five standard mixings were prepared. The phosphate and calcium contents of the “blank feed’’ were obtained by the proposed The dicalcium orthophosphate was first dissolved in hydrochloric acid before procedures.aliquots were taken for the determination. PKOPOSED PROCEDURE FOR PHOSPHATE DETERMINATION REAGENTS- Ammonium va?zadomoZybdate compZex3-Dissolve 40 g of ammonium molybdate in 400 ml of water at 50” C. Dissolve 1 g of ammonium vanadate in 300 ml of boiling water. Cool, add to the solution 200 ml of AnalaR nitric acid. Mix both solutions together and make up the volume to 1 litre. Standard Phosphate sohiion-Dissolve 0.4394 g of potassium dihydrogen orthophosphate, KH,PO,, in 1 litre of distilled water. (1 ml of solution == 0-2 mg of phosphorus pentoxide.) CALIBRATION OF GRAPH FOR PHOSPHORUS PENTOXIDE- Transfer by pipette 5, 10, 15, 20 and 25-ml aliquots of the standard phosphate solution into 100-ml calibrated flasks.Add 30ml of the ammonium vanadomolybdate reagent to each flask in turn with continuous shaking of the flask. Make the solution up to the mark with distilled water. Allow the flasks to stand for 1 to 2 hours. Measure the optical density at 436mp with a Unicam SP600 operated at maximum sensitivity (or any other similar instrument), against a distilled water - reagent blank. Draw a graph of optical densities against milligrams of phosphorus pentoxide. DETERMINATION- Transfer by pipette 5 ml of the Kjeldahl-digestion solution” into a 100-ml calibrated flask. Add 1 to 2 drops of phenolphthalein and make the solution just alkaline with 30 per cent. * This solution is obtained from the normal Kjeldahl digestion, with a catalyst containing potassium sulphate and copper sulphate; the digest is transferred to the calibrated flask by washing with distilled water.108 STUFFINS : DETERMINATION OF PHOSPHATE [Analyst, Vol.92 sodium hydroxide; then, with 10 per cent. sulphuric acid adjust the solution to neutral or faintly acidic. Add 30 ml of vanadate reagent (shake the flask continuously during addition). Make up to the 100-ml mark with distilled water. Allow the solution to stand for 1 to 2 hours and measure the optical density at 436mp. PROPOSED PROCEDURE FOR CALCIUM DETERMINATION REAGENTS- Potassium permanganate, N. Ammonia solution, 50 per cent. v/v. Sulfihuuric acid, 8 per cent. v/v. Hydrochloric acid, 2 N. Ammonium oxalate, (saturated a d hot). Methyl red.PROCEDURE- Add about 50ml of distilled water and 2 to 3 drops of methyl-red solution. Make the mixture just alkaline with 50 per cent. ammonia solution, then by adding 2 N hydrochloric acid just acid again. Dilute the solution to about 150 ml with distilled water, and bring it to the boil. While stirring add slowly 10ml of hot saturated oxalate solution. If the solution becomes yellow - orange add 2 N hydrochloric acid until it becomes pink. Allow to stand for 1 hour. Filter the mixture through a Whatman No. 44 filter-paper. Wash the filter-paper with warm water. After dissolving the precipitate in 8 per cent. sulphuric acid carry out the normal procedure for determining calcium by titration with permanganate. When these procedures were applied to the “blank feed’’ and dicalciuni orthophosphate they were found to contain- Transfer by pipette 40 ml of the Kjeldahl-digestion solution into a 250-ml beaker.Phosphorus Calcium oxide, pentoxide, per cent. per cent. “Blank feed” . . .. .. . . 0.35 0.18 Dicalcium orthophosphate . . .. 30.35 34.6 The five standards thus contained the following amounts of added and total phosphate and calcium- Added Total ----A- 17------7 Calcium Phosphorus Calcium Phosphorus Standard oxide pentoxide oxide pentoxide A 0.30 0.34 0.65 0.52 B 0.595 0.678 0.945 0.858 C 1.167 1-33 1.517 1.51 D 1-718 1.96 2-068 2-14 E 6.0 6-85 6-35 7.06 PHOSPHATE DETERMINATION EXPERIMENTS- Four procedures were followed in the determination of phosphate in the five standard samples by combining two methods of sample preparation, vix., dry-ashing and Kjeldahl digestion, with two finishes : colorimetric determination with vanadate reagent,3 and with reduced molybdate reagent .4 95 $6 The results shown in Table I indicate that lower recoveries are obtained with the dry- ashing technique.The possibility of losing phosphoric acid when dry-ashing has been known generally for some considerable time. It is suggested in the Fertiliser and Feeding Stuffs Regulations 1955, that a nitrate should be added before incineration of the sample, to avoid loss of phosphoric acid.February, 19671 AND CALCIUM IN FEEDING STUFFS TABLE I THE EFFECT OF TWO METHODS OF SAMPLE PREPARATION 109 (a) Kjeldahl digestion- Vanadate P,O, found, Recovery, Standard per cent. per cent. A r 1 A 0.49, 0.50, 0.52 96.6 B 0.83, 0-85, 0.87 99.1 C 1.42, 1.40, 1.47 94-6 D 2.05, 2.03 95.3 E 6-88, 6.86 97.3 (b) Dyy-ashing- Vanadate , 3 P,O, found, Recovery, Standard per cent.per cent. A 0.48, 0-48 91-7 B 0.62, 0-73, 0-73 80.8 C 1.22, 1-15, 1.27 80.1 D 1.76, 1.56, 1.67 77.6 E 6.86, 6-90 97-4 Reduced molybdate P,O, found, Recovery, per cent. per cent. 0.53, 0-56 104-8 0.88, 0-90 103.7 1-51 100.0 2.12, 2.08 98.1 6-64, 6.68 94.3 A 7 7 Reduced molybdate P,O, found, Recovery, per cent. per cent. 0.51, 0.47 94-2 0.73, 0.72 84.5 1-21, 1.18 79.5 1.58 73.8 6-62, 6.62 93-8 r A \ Some experiments were carried out to ascertain the effect of adding an oxidising agent before dry-ashing, on the loss of phosphoric acid. This technique adopted here may not have been suitable because the recoveries of phosphoric acid were by no means satisfactory.(See Table 11). However, an interesting observation in connection with the loss of phosphoric acid on dry-ashing was obtained later. (See Table I11 and the comments). TABLE I1 THE EFFECT OF TWO METHODS OF OXIDATION BEFORE DRY-ASHING ( a ) Addition of nityic acid- Vanadate Reduced molybdate A I > f A \ P,O, found, Recovery, P,O, found, Recovery, Standard per cent. per cent. per cent. per cent. C 1.58, 1-44 100.0 1-15, 1.26 79.5 D 2.08 97.2 1.47, 1.54 70-6 (b) Addition of fiotassium nityate- Vanadate Reduced molybdate r----A-- 7 7-p P,O, found, Recovery, P,O, found, Recovery, Standard per cent. per cent. per ccnt. per cent. A 0.51 98.1 B 0-77 89.7 C 1.32 87.4 D 1.78 84.1 0.4G 0.74 1-15 1.34 88.4 86.2 7G.1 62.6 It will be seen from Table I that reasonably accurate results are obtained when the Kjeldahl-digestion solution is used for the phosphate determination either by the vanadate or reduced molybdate procedures.Whereas the vanadate procedure produces slightly low results (96 per cent.) the reduced molybdate figures are slightly high (101-6 per cent.). Because the vanadate procedure requires less manipulative work and involves less time, it is the recommended technique. To obtain a more comprehensive picture of the variations expected by the adoption of the recommended procedures, further work was carried out on samples containing various levels of calcium and phosphate.110 STUFFINS DETERMINATION OF PHOSPHATE [Analyst, Vol. 92 The following levels of phosphate and calcium were taken- (a) Four poultry food samples containing 4 to 5 per cent.of calcium oxide and 1.5 to 2.0 per cent. of phosphorus pentoxide (laying foods). (b) Four poultry food samples containing 2 to 3 per cent. of calcium oxide and 1.5 to 2.0 per cent. of phosphorus pentoxide (growing foods). (c) Two samples of fish meal containing about 6 per cent. of calcium oxides and about 6 per cent. of phosphorus pentoxide. (d) Two samples of meat meal containing about 9 per cent. of calcium oxide and about 7 per cent. of phosphorus pentoxide. The results of this work are shown in Table 111. TABLE I11 DETERMISATION OF VARIOUS LEVELS OF CALCIUM AND PHOSPHATE FOR STANDARD AKALYSIS Kjeldahl digestion Dry-ashing Vanadate Molybdate Vanadate Mol ybdate A r A 7 r 7 Sample CaO P20, P20, P20, P205 CaO P205 P205 P205 P205 A { i::; 1.58 1.60 1-69 1.62 { t::: 1.58 1.69 1.61 1.76 L { ::? 1.85 1.85 1-95 1.90 {::!: 1.83 1.84 1.97 1.99 11 { E::8 1-82 1.77 1-94 1-83 {:::: 1.89 1.88 1.97 1-91 S {::is 1-50 1.54 1.59 1-53 {::A: 1-51 1-49 1.63 1.71 40 { i::: 1-87 1.83 1-92 1.83 { i:;: 1.73 1-78 1.51 1.60 3743 { t:;: 1.97 1.91 1.98 1.88 { i::; 1.76 1.82 1.54 1-58 3744 {E::! 1-65 1-69 1-69 1-70 { i::: 1.63 1.60 1.50 1.54 3746 { i::: 1.66 1.66 1.61 1-71 {E:; 1.64 1.64 1-56 1.56 Xeat meal { ::i: 6-70 6-78 6.74 7.10 { i::: 6.70 6.40 6.72 6-98 Meat meal { :::: 6.56 6.99 6.50 7-18 { ::i6 6.70 6.56 6.86 7.08 Fish meal { :::: 5.90 6.01 5.9G 6.06 { :::! 5-80 5.70 5.68 5.98 Fish meal { :::: 5-68 5.60 5.90 5.83 { ::;: 5.68 5.64 5.68 6-14 The following statistical analysis was obtained from the results in Table III- Calcium determination- Kjeldahl digestion (means) Material KD Growing foods .. . . 2.29 Animal protein.. . . 7.09 Laying foods . . .. 4.47 P7aosphate determination- Kjeldahl digestion (means) Material KD Laying foods . . . . 1.72 Crowing foods . . .. 1.78 -Animal protein.. .. 6.34 Least Dry- significant ashing difference (means) (mean of 8) D h p = 0.05 Significance 4.39 0.19 Not significant 2.51 0-19 Significant (p = 0.05) KD < DA 7.54 0.19 Significant (p = 0.01) KD < DA Least Dry- significant ashing difference (means) (mesnof 16) DA p = 0.05) Significance 1-77 0.09 Not significant 1.62 0.09 Significant (p=O.Ol) DA < KD 6.26 0.09 Not significantFebruary, 19671 AND CALCIUM IN FEEDING STUFFS 111 In Table I11 further confirmation is obtained of the observation made earlier in this paper with reference to the loss of phosphoric acid on dry-ashing (Table I).The samples 40, 3743, 3744 and 3746 contain similar amounts of calcium and phosphoric acid as the standards C and D of Table I where similar results were obtained. However, the other results in Table I11 do not indicate any loss of phosphoric acid when the calcium content is at least twice that of the phosphoric acid. The vanadate procedure can tolerate 1000 p.p.m. of copper and 100 p.p.m. of iron without interference. Neither of these two elements is likely to be present to this extent in the aliquot taken for the determination. Manganese and zinc do not interfere. With the calcium determination, however, we have the reverse effect of that which takes place with phosphate.For samples 40, 3743, 3744 and 3746 higher recoveries are obtained on dry-ashing. This latter observation is further illustrated in the investigation of the recommended method for calcium, in which a similar pattern of work was carried out on the standards to that adopted for the phosphate. These results are shown in Table IV. TABLE IV THE EFFECT OF TWO METHODS OF SAMPLE PREPARATION (a) ICjeldahl digestion 7- - I CaO found, Recovery, Sample per cent. per cent. A 0.65, 0.64 99.2 B 0.98, 1.0, 0.92 102.3 C 1.50, 1-50 98.9 D 2-08, 2.03, 2-16 101.1 E 6.06, 6.14 96.1 (b) Dry-asliing CaO found, Recovery] per cent. per cent. 0.69, 0.71, 0.71 106-1 0.99, 1.02 106.3 1-55, 1.57 102.8 2.11, 2-13 102-5 6.41, 6.48 101.5 The adoption of the recommended technique enabled these laboratories to carry out the determination of protein, calcium and phosphate on the same Kjeldahl-digest solution. The protein determination required only a small aliquot of the digest solution, and, conse- quently, sufficient solution remains for the determination of calcium and phosphate. The gravimetric method for phosphate has not been considered in this work because the colorimetric procedures are much less tedious but equally accurate. The adoption of the recommended procedures enabled the 2-fold aim of the investigation to be achieved, namely: speed-up of analyses; and elimination of the need of apparatus such as muffles and crucibles. I thank the Directors of J. Bibby & Sons Ltd. for their permission to publish this paper. REFERENCES 1. 2. 3. 4. 5. 6. Pulss, G., Landw. Forsch., 1961, 14, 38. - , 2. analyt. Chem., 1960, 176, 412. Kitson, R. E., and Mellon, M. G., Ind. Engng Chem. Analyt. Edn, 1944, 16, 379. Finzade, C. H., Ibid., 1935, 1, 227. Gerritz, H. W., J . Ass. Off. Agvic. Chem., 1940, 23, 321. Schricker, J., and Duncan, I?., Ibid., 1939, 22, 167. Received June 1st. 1966