Digesting Biological Materials for Calcium and Phosphorus Analysis H. W. GERRITZ, Division of Chemistry, Agricultural Experiment Station, Pullman, Wash.
I
T IS OFTEN necessary to make both calcium and phos-
phorus determinations on biological materials, particularly in vitamin D studies where the calcium and phosphorus ratio is of importance. In most cases it has been customary to prepare samples separately for the determination of the two elements, especially where the percentage of phosphorus in a material is high. A rapid method of removing organic matter which will permit the determination of both calcium and phosphorus in the digest permits a saving of time on the part of the analyst and a saving of equipment and reagents to the laboratory. The following procedure is adapted to plant and animal material and has been found to give excellent results on such materials as feed concentrates, feces, and grasses. It should also be possible to determine mineral elements such as iron, magnesium, and others not forming insoluble perchlorate salts on this digest.
Procedure Weighed samples of suitable size are placed in 500-ml. Kjeldahl flasks. Twenty to 30 ml. of concentrated nitric acid are added and the flasks are placed on asbestos gauzes over medium flames. The contents are boiled gently with frequent mixing until the samples pass into a semi-colloidal solution. The particles of material become swollen and gel-like, then disintegrate, producing a finer suspension or a solution. Experience will indicate at what time this occurs, and the speed of the digestion may be accelerated materially by determining the length of time required for the material being analyzed to reach this stage. For the materials reported in this paper the flames were so adjusted that the treatment took 30 to 45 minutes. Heating to dryness must be avoided. Ten milliliters of 70 per cent perchloric acid are now added to each and the flasks are placed over free flames. Very low flames are necessary during the perchloric acid oxidation; best results are obtained when just sufficient heat is applied to keep the solution boiling. Only a fine point of the flame should impinge on the flask. Higher temperatures tend to drive off the perchloric acid without materially accelerating oxidation. When fuming begins, the flame is so adjusted that only a trace of the perchloric acid fumes reaches the u per region of the Kjeldahl neck. The heating is continued untifthe solution is practically colorless or only a faint yellow color remains. The solution is allowed to cool slightly and 50 ml. of distilled water are added. Vigorous boiling occurs which drives out the remaining nitrogen dioxide fumes, leaving a clear solution. The solution is filtered into a volumetric flask, and the Kjeldahl is thoroughly washed with distilled water. When the solution has cooled it is made to volume and aliquots are taken for analysis. Calcium and phosphorus determinations may be completed by the usual procedures.
Results Data reported have been obtained by official methods (1).
TABLEI. CALCIUM DETERMINATIONS Digested wi$h Nitric and Perchloric Acids A B Diff. Av.
%
%
%
€3
Dlff.
Av.
Diff. between Acid Digestion andAshing
%
%
%
%
%
In Pasture Grass Volumetric Method 0.94 1.03 0.98 1.07 0.85 0.87 0.52 0.57 0.46 0.34
0.93 1.00 0.98 1.08 0.85 0.89 0.53 0.59 0.48 0.33
0.01 0.03 0.00 0.01 0.00 0.02 0.01 0.02 0.02 0.01
0.67 0.73
0.72 0.72 3.89 3.45 3.56 3.34 3.57 3.34 3.23 3.74 3.27 3.39
0.05 0.01 0.02 0.03
0.94 1.02 0.98 1.08 0.85 0.88 0.53 0.58 0.47 0.34
0.93 1.03 0.97 1.06 0.87 0.88 0.48 0.57 0.48 0.34
0.93 1.04 0.99 1.08 0.88 0.84 0.48 0.56 0.48 0.34
0.00 0.01 0.02 0.02 0.01 0.04 0.00 0.01 0.00 0.00
0.93 1.04 0.98 1.07 0.88
0.86
0.48 0.57 0.48 0.34
10.01 -0.02 0.00 +0.01 -0.03 $0.02 +0.05 +0.01 -0.01 0.00
I n Poultry Feces, Volumetrio Method 3.87
3.48 3.56 3.29 3.56 3 38 3.24 3.74 3.29 3.34
0.70 0.73
3.88
3.47 3.56 3.32 3.57 3.36 3.24 3.74 3.28 3.37
0.00
0.05 0.01 0.04 0.01 0.00 0.02 0.05
0.73 0.67 3.89 3.48 3.51 3.38 3.61 3.20 3.20 3.81 3.25 3.38
0.69 0.70 3.82 3.48 3.63 3.38 3.57 3.38 3.25 3.79 3.25 3.49
0.04 0.03 0.07 0.00 0.02 0.00 0.04 0.18 0.05 0.02 0.00 0.11
0.71 0.69 3.86 3.48 3.52 3.38 3.59 3.29 3.23
3.80 3.25 3.42
-0.01 f0.04 +0.02 -0.01 +O. 04 -0.06 -0.02 +0.07 +o. 01 -0.06 +0.03 -0.05
In Poultry Feeds, Volumetric Method 1.48 2.27 1.98 1.90 1.70 2.44 0.29 0.21 0.20 0.25 3.23
1.48 2.30 2.00 1.94 1.73 2.44 0.27 0.26 0.25 0.25 3.24
0.00 0.03 0.02 0.04 0.03 0.00 0.02 0.05 0.05 0.00 0.01
1.48 2.29 1.99 1.92 1.72 2.44 0.28 0.24 0.23 0.25 3.24
1.48 2.28 1.98 2.00 1.86 2.42 0.20 0.22 0.24 0.26 3.17
1.50 2.24 1.94 1.99 1.88 2.46 0.21 0.26 0.28 0.27 3.19
0.02 0.04 0.04 0.01 0.02 0.04 0.01 0.04 0.04 0.01 0.02
1.49 2.26 1.96 2.00 1.87 2.44 0.21 0.24 0.26 0.27 3.18
-0.01
+0.03 +0.08 0.00 -0.09 0.00 4-0.06 0.00 -0.03 -0.02
+0.06
TABLE 11. PHOSPHORUS DETERMINATIONS Aliquot3 from Solutions Prepared for Calcium A B Diff. Av.
%
%
%
%
Digested with Hydrochlorio and Nitric Acids A B Diff. Av.
%
%
%
%
Difference in Two Methodp
%
In Pasture Grass, Volumetric Method 0.50 0.35 0.44 0.48 0.46 0.44 0.63 0.67 0.58 0.51
0.50 0.35 0.44 0.48 0.47 0.43 0.63 0.67 0.59 0.52
0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.01
0.98 1.08 1.53 1.50 1.48 1.49 1.59 1.52 1.51 1.62 1.68 1.61
0.98 1.10 1.54 1.50 1.51 1.48 1.59 1.52 1.49 1.57 1.66 1.61
0.00 0.02 0.01 0.00 0.03 0.01 0.00 0.00 0.02 0.05 0.02 0.00
0.50 0.36 0.44 0.48 0.47 0.44 0.63 0.67 0.59 0.52
0.59 0.35 0.47 0.45 0.45 0.44 0.55 0.64 0.56 0.53
0.59 0.00 0.59 0.39 0.04 0.37 0.47 0.00 0.47 0.44 0.01 0.45 0.44 0.01 0.45 0.44 0.00 0.44 0.54 0.01 0.55 0.64 0.00 0.64 0.55 0.01 0.56 0.53 0.00 0.53
-0.09 -0.02 -0.03 -0.03 +o. 02 0.00
+0.08 +0.03 +0.03 -0.01
In Poultry Feces, Gravimetric Method 0.98 1.09 1.54 1.50 1.50 1.49 1.59 1.52 1.50 1.60 1.67 1.61
0.96 1.06 1.51 1.49 1.49 1.52 1.49 1.57 1.60 1.55 1.60 1.52
0.94 1.11 1.51 1.55 1.46 1.54 1.63 1.60 1.54 1.59 1.66 1.53
0.02 0.05 0.00 0.05 0.03 0.02 0.14 0.03 0.06 0.04 0.06 0.01
0.95 1.09 1.51 1.52 1.48 1.53 1.56 1.58 1.57 1.57 1.63 1.63
$0.03 0.00
+0.03 -0.02 +0.02 -0.04 f0.03 -0.06 -0.07 f0.03 +0.04 f0.08
In Poultry Feeds, Gravimetric Method Digested with Nitric and Sulfuric Apids and Sodium Nitrate
In the comparisons reported duplicate samples were digested by the proposed method and a t the same time duplicate samples were prepared according to official methods. The comparisons are given in Tables I and 11. A plus sign indicates that results by the proposed method are higher, while a minus sign indicates that they are lower than the results by official methods of preparing the sample. A and B indicate duplicate digestions. Results by the two methods agree closely and the differences are essentially the same as differences between duplicates by the same digestion method.
%
-Ashed,A
0.52 0.51 1.01 0.99 0.97 0.98 1.00 1.00 1.00 0.99 1.11 1.05 1.18 1.17 0.57 0.56 0.61 0.61 0.66 0.61
0.56 2.30
167
0.01 0.02 0.01 0.00 0.01 0.03 0.01 0.01 0.00 0.04 0.66 0.00 2.29 0.01
0.52 1.00 0.98 1.00 1.00 1.10 1.18 0.56 0.51 0.53 0.55
2.30
0.52 0.96 0.92 0.97 0.92 1.06 1.12 0.61 0.63 0.61 0.62 2.25
0.58 0.84 0.91 0.95 0.87 1.11 1.14 0.61 0.69 0.61 0.60 2.16
0.06 0.12 0.01 0.02 0.05 0.05 0.02 0.00 0.06 0.10
0.02
0.09
0.56 0.90 0.92 0.96 0.90 1.09 1.13 0.61 0.56 0.56 0.61 2.21
-0.04 +0.10 4-0.06 t0.04 +0.10 +0.01 +0.05 -0.05 -0.05
-0.03
f0.04 +0.09
168
INDUSTRIAL AND ENGINEERING CHEMISTRY
Discussion Four-gram samples of pasture grass were digested in order to get sufficient calcium and phosphorus for the determinations. The time for digestion was about 1.5 hours. Two-gram samples of feed and feces were digested in 1.25 hours. When large amounts of potassium were present in the sample, more perchloric acid was required because of precipitation of potassium perchlorate. The digestion is not difficult to conduct, but care must be taken that the sample is not boiled to dryness. Maximum speed of digestion will be obtained by noting carefully the time reauired for the nitric acid to emulsify the material. Insufficient heat tends to make the digestion lengthy, while excess heat boils off the nitric acid before its maximum effect the nitric k obtained. It is not advisable, therefore, to acid over a free flame. Best results, together with rapid digestion, were obtained by placing the flask on an asbestos
VOL. 7, NO. 3
gauze over a gas flame in a Kjeldahl digestion rack and turning on a full flame. The digestion produces a clear solution in a short time, thus effecting a saving of time and apparatus when both calcium and phosphorus are to be determined on the same material. It is much more rapid than ashing and may therefore prove more efficient even when calcium or other elements alone are to be determined. Iron, magnesium, and other elements not forming insoluble perchlorate salts might also determined on the digest.
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods, 3rd ed., 1930. with R~~~~~~~ ~~~~h 22, 1936. hi^ work was begun by the author the Division of Dairy Husbandry. Soientifio Paper No. 310,College of Agriculture and Experiment Station, State College of Washington.
Impurities in White Sugars VIII. Effects of Some Impurities on Decomposition of Sucrose during the Barley Candy Test J. A. AMBLER and S. BYALL, Bureau of Chemistry and Soils, Washington, D. C.
‘
I
Results obtained in the barley candy test for quality of sugar, a detailed description of which is given, are subject to variation due to slight differences in the routine of different operators, to differences in rate and duration of heating, and to different thermal properties of different pieces of apparatus. However, the results of a series of tests made by an experienced operator using the same utensils are comparable among themselves. The effects of various salts and organic substances which may be present in white sugars were studied by making the candy test on a very pure standard sugar to which the nonsugar was added in known proportions. From the analyses of these candies it is seen that the nonsugars fall into three major groups : those of Group I increase the inversion of sucrose but inhibit caramelization or are without effect on either; those of Group I1 tend to inhibit inversion but increase the amount of caramelization;
T HAS long been a matter of general experience, especially among manufacturers of food products, that some commercial white sugars, when subjected to the elevated temperatures required in many processes, withstand the heating with less discoloration or with the formation of less invert sugar than others. Therefore there arose the practice of testing samples of new or prospective purchases of sugar (25) by quick, practical tests, the conditions of which approximate those to which the sugar would be subjected
and those of Group I11 increase both the inversion and degree of caramelization. The auto-inversion of sucrose and the different effects of the nonsugars on the decomposition of sucrose during the candy test are explained by evidence showing that sucrose acquires an enhanced acidic nature at high temperatures and, together with its decomposition products, reacts with the nonsugars present in the sirup, causing greater inversion and less caramelization when the resultant hot sirups are acidic in reaction and less inversion and greater caramelization when they are alkaline in reaction. The increase in both inversion and caramelization in the presence of the nonsugars of Group 111, which are acidic in reaction, is due to secondary reactions of the particular nonsugar with the invert sugar and other degradation products formed from the sucrose during the test. Salts of volatile or unstable acids react as alkaline salts. during the making of the finished food product. The cause of such variations in stability of different lots of sugar is ascribed to the effects of the impurities, or nonsugars, contained in small amounts in the white sugar, and hence it becomes of interest to know the effects of the commonly occurring nonsugars on the heat-resisting qualities of sucrose. The practical tests developed for this purpose are of two types, the caramelization test, in which the dry sugar kt heated, and the so-called “barley candy” test, in which 5
