Determination of Blood in Packing-House
By-products RAYMOND REISER
AND
G. S. FRAPS, Texas Agricultural Experiment Station, College Station, Texas
T
HE determination of the blood content of meat products
550 mp between the reduced and unreduced solutions are given in Table I, as averages of two determinations. The density is in proportion to the concentration, within the limits of experimental error. Ten different samples of dried blood were secured from Swift and Company, Armour and Company, and Wilson and Company, whose cooperation is hereby acknowledged. The density of the color derived from hemoglobin was determined by the method described below, with the results given in Table 11. The relative dried blood value is the density of each sample divided by the mean density and multiplied by 100. The hemoglobin was calculated by the method described below. The density of the color of 0.1 per cent solutions of the various samples ranges from 0.39 to 0.71, showing a wide variation in the hematin content. The mean is 0.55, with a standard deviation of 0.10. The mean density of 0.55 was used for the calculation of dried blood in dried animal by-products. The comparative dried blood content (Table 11) ranges from 71 to 129. These differences are probably due partly to the method of drying, since this affects the hemoglobin which remains in the dried blood. The method of preparing the tankage or meat scraps would have some effect upon the quantity of dried blood found by this method.
used in animal feeding was undertaken as part of a study of the various constituents of commercial packing-house byproducts. As hemoglobin is the chief constituent of blood, the determination of hemoglobin or its derivatives was used to estimate the quantity of dried blood present. Experiments with fresh blood showed that the quantity of hemoglobin in the dried product depends to some extent upon the method of processing. This must be considered in the interpretation of the results of the analyses.
Development of Method for Dried Blood The determination of dried blood in tankage and meat scraps was based upon the hemoglobin content of commercial dried bloods. Hemoglobin in fresh blood is usually determined by diluting it with dilute hydrochloric acid and reading the color against a standard colored disk. This method is not applicable to dried blood because hematin dissolved in alkali precipitates when acid is added and the color cannot be read. Dried blood is readily dissolved by boiling in 1 per cent sodium hydroxide, but the color of the solution is not proportional to the amount of hemoglobin because of the presence of other colored matter. A suitable method was found to be reduction with sodium hydrosulfite in the presence of pyridine, an alkaline pyridine hemochromogen being produced similar to that described by Drabkin and Austin (2). Portions of the reduced and unreduced solutions were placed in opposing cups and the difference in color a t 550 mp, the wave length of maximum absorption, was read in terms of density by means of a Bausch & Lomb visual spectrophotometer. Dried blood did not dissolve well in solutions of sodium phosphates, ammonia, or dilute acids. It was thought that boiling in acid might change the dried blood, so that upon making the boiled mixture slightly alkaline the blood would dissolve, but 0.05 to 1.00 per cent hydrochloric acid and buffered solutions from pH 3 to 10 did not have this effect. The maximum amount of color of a 0.1 per cent of solution of dried blood (density 0.51) was produced with 0.5 and 1.0 per cent sodium hydroxide. A smaller amount of color (density 0.46) was obtained with 0.2 per cent and still smaller (density 0.21) with 0.05 per cent sodium hydroxide. Approximately 20 minutes were required to dissolve 0.1 gram of dried blood in 100 ml. of boiling 1 per cent sodium hydroxide. Some samples dissolved in less time. When a sample of dried blood was boiled in 1 per cent sodium hydroxide for different periods of time the density of the color was 0.65 when boiled 20 minutes, 0.59 when boiled 40 minutes, and 0.51 when boiled 60 minutes. The hematin is destroyed by continued boiling, so that the time of boiling should be held to the 20 minutes necessary for complete solution. Drabkin and Austin (2) used a 30 per cent pyridine solution for the development of the hemochromogen. Because of the large number of determinations planned for the present work, tests were made to see if less pyridine could be used. Five per cent pyridine was found to be sufficient, since 5, 10, 20, and 30 per cent produced the same quantity of color. The relation of the concentration of blood to the absorption of light by the color produced was studied with 0.2 gram of a commercial dried blood dissolved in 100 ml. of 1 per cent sodium hydroxide and 5 per cent of pyridine, diluted to various concentrations. The differences in density of color a t
Development of Method for Hemoglobin I n the work discussed above, comparisons were made with commercial dried bloods and not with hemoglobin. I n order to ascertain the relation of the modified hemoglobin of commercial blood to hemoglobin, work was done with fresh cow blood. These samples were furnished by H. Schmidt of the Veterinary Division of the Experiment Station.
TABLEI. RELATION BETWEEN CONCENTRATION OF BLOOD SOLUTION AND DENSITY OF COLOR PRODUCED Concentration of Blood
Density of Color
Density per 1%
0.11 0.22 0.32 0.42 0.52 0.62 0.72 0.83 0.95 1.06
5.5 5.5 5.3 5.3 5.2 5.2 5.1 5.2 5.3 5.3 5.3
70 0.02 0.04 0.06 0.05 0.10 0.12 0.14 0.16 0.18 0.20
Mean
TABLE11. HEMOGLOBIN AND RELATIVE DRIEDBLOOD CONTENT OF COMMERCIAL DRIEDBLOODS Density of Color
Relative Dried Blood Value
% 0.46 0.64 0.42 0.60 0.56
Av.
851
0.58 0.56 0.39 0.56 0.71 0.55
84 116 76 109 102 105 102 71 102 129 100
Hemoglobin % 48 67 44 63 59 61 59 41 59 75 58
852
Vol. 14, No. 11
INDUSTRIAL AND ENGINEERING CHEMISTRY
TABLE111. EFFECTOF HEATINGAND PRESSURE UPON HEMOGLOBIN (As measured by density of color produced) Wave Density at Length Maximum Time Maximum Wave Treatment Treated Absorption Length Hours mu None 0 564 1.17 Autoclaved at 15 pounds' 0.5 550 0.64 pressure 1 550 0.45 1.6 550 0.33 2 550 0.26 3 550 0.19 Aeated at 200' in dry oven 1 650 0.41
Density at 550 m# 0.78 0.64 0.46 0.33 0.26 0.19 0.41
When fresh blood was dried at 100" C., pulverized and dissolved in alkali and the hemochromogen was developed] the wave length of maximum absorption was near 562 mp, while that of the hemochromogen of commercial dried blood was 550 mp. The processing changed the hemochromogen. In order to find the conditions required to convert the hemoglobin of fresh dried blood to the same substance as that of commercial dried blood, so that the two could be compared, 100mg. samples of fresh dried cow's blood were heated to 200" C. in a dry heat or autoclaved for various lengths of time a t 6.8 kg. (15 pounds) pressure and the color of the resulting hemochromogen was measured. The results, given in Table 111, show that all these treatments produced a hemochromogen with a wave length of maximum absorption of 550 mp, Autoclaving longer than 0.5 hour destroyed progressively the hemochromogen-forming powers of the blood, since the density of the color decreased with the time of autoclaving. It is evident that the hemochromogen-forming powers of commercial dried blood must similarly depend upon the temperature of drying or other treatment. This may be the cause of some of the differences in commercial dried blood shown in Table I. The relation between the hemoglobin of fresh blood and that of dried blood was studied on five samples of fresh cow blood. Hemoglobin wm determined by the acid hematin method of Newcomer (€4,by means of the Bausch & Lomk s ectrophotomein a vacuum ter. Water was determined by drying at 100 oven. The dried bloods from the water determination were ground in a mortar and amounts containing 200 mg. of hemolobin autoclaved for 30 minutes at 6.8 kg. (15 pounds) ressure, $issolved in 1 per cent sodium hydroxide, 5 cc. of pyri8ne were added, and made up to 100 ml. The resulting 0.20 per cent hemoglobin solutions were filtered and diluted to 0.05, 0.10, and 0.15 per cent. Ten milliliters of each dilution were reduced with a few milligrams of sodium hydrosulfite, and, after 10 minutes the difference in density a t 550 mp between the reduced and unreduced solutions was determined in opposing cups of the spectrophotometer.
8.
The average density for 1 per cent hemoglobin for four of the samples is 9.5 (Table IV). The density is not quite constant a t the different dilutions but does not change regularly with concentration, differences a t different concentrations apparently being due to experimental error. The extinction coefficients given in Table IV were calculated from the equaD tion c = e + in~ which c is the percentage concentration of hemoglobin, D is the difference in density between the reduced and unreduced solutions, e is the extinction coefficient, and L is the depth of the solution in cm. I n four of the five samples the mean extinction coefficients were 4.79, 4.61, 4.86, and 4.78. I n the fifth blood it was 3.54. In the preparation of commercial dried blood, the fresh blood is usually coagulated, drained, and dried a t a high temperature (3). The five samples of fresh blood contained 70.2 to 77.7 per
cent hemoglobin with a mean of 73.6, calculated to the dry basis, but commercial dry blood contained only 58 per cent (Table 11). Two of the nine commercial dry bloods contained hemoglobin within the range of the fresh bloods-namely, 67 and 75 per cent, respectively. The low hemoglobin content of the other samples may have been due to the method used in drying the blood. Another incongruity is the low extinction coefficient of the color developed from one of the fresh bloods. In four samples these values were 4.79, 4.86, 4.61, and 4.78 (Table IV) but in a fifth it was 3.54. This blood had a low hemoglobin content of only 10.8 per cent as compared with 13.6, 13.9, 14.5, and 15.0 per cent for the other samples, yet on the dry basis the hemoglobin content was similar to that of the other samples. It would appear that the hemoglobin of this blood was more easily destroyed by heat than that of normal blood. Since the hemoglobin in blood is affected both by the time and method of heating, it may be partly destroyed in the manufacture of tankage or meat scraps and the percentage destroyed will depend upon the treatment given. The results secured by the method here described, are more likely to be too low than too high and are not highly accurate.
Determination of Dried Blood or Hemoglobin Extraction of meat scraps or tankage with ether was necessary to remove substances which cause a cloudy filtrate. One gram of meat scraps or tanka e is washed four times on a S. P. ether, or is placed filter paper with 10-ml. portions of in a centrifuge tube, 10 ml. of ether are added, allowed to stand 10 minutes, and centrifuged, and this treatment is repeated twice. It is then ground in a mortar, transferred to a 400-ml. beaker with 100 ml. of I per cent sodium hydroxide, and gently boiled for 20 minutes. The solution, now about 75 ml., is transferred to a 100-ml. volumetric flask containing 5 ml. of pyridine, made up to volume, with distilled water and filtered. A few milligrams of sodium hydrosulfite are added to 10 ml. of the filtered solution in a test tube. After standing not less than 10 minutes and not more than 30 minutes, portions of the reduced and unreduced solutions are placed in opposite cups of the s ectrophotometer. The density, read at 550 mp and 2-cm. e th, is the difference in density between the reduced and unre uced hemochromogen, and is read directly from the instrument. When the quantities and dilutions given above are used, the percentage of dried blood is obtained by dividing the density as read by 5.5, the average density of a 1 per cent solution of com-
6.
aIp
TABLE IV. RELATION OF CONCENTRATION OF HEMOGLOBIN TO DENSITYAND EXTINCTION COEFFICIENT OF COLORPRODUCED Laboratory
KO.
Concentration of
Hemoglobin, %
Density 2 Cm.
Extinction Coefficient
Density for 1% Hemoglobin
0.10 0.15 0.20 64,345
64,346
64,347
64.348
0.05 0.10 0.15 0.20
0.34 0.67 1.05 1.57
3.40 3.85 3.50 3.43 Mean 3.54 0.05 0.44 4.40 0 10 0.88 4.40 0 15 1.48 4.90 0 20 1.89 4.73 ,Mean 4.61 0.05 0.46 4.60 0.10 0.98 4.90 0 15 1.49 4.91 0.20 2.04 5.01 Mean 4.56 0.05 0.43 4.30 0.10 0.99 4.95 0.15 1.45 4.87 0.20 1.03 5.01 Mean 4.78 Grand mean, 64,345excluded 4.76
6.8 6.7 7.0 6.9 8 8 8.8 9 9 9.0 9 2 9 8 10 0 10.2 8.6 9.9 9.8 10.I 9.5
ANALYTICAL EDITION
November 15, 1942
Hemoglobin %
Meat meal and meat scraps, as defined by the Association of American Feed Control Officials ( I ) , should not contain blood beyond such traces as might occur unavoidably in good factory practice, while blood is not excluded from tankage. According to Kraybill (4) blood is usually present in tankage.
18.1 17.8 18.1
Summary
AND HEMOGLOBIX IN MEATBYTABLEV. DRIEDBLOOD PRODUCTS
Dried Blood
Deecription
% Tankage
60% protein digester tankage
Tankage 60% protein digester tankage 60% protein digester tankage with bone 60% protein digester tankage Meat and bone scraps
30.8
30.6 31.0 27.0 20.2 11.9
__ .
41 B -
5.2 0.9
50% protein meat and bone scraps
Meat and bone scraps
Steamed bone meal 20% protein steamed bone meal 65% proteln sardine meal
3.1 2.3 2.3 3.2 1.1 4.1 1.4
26.5
1.3 12.8 0
0.5 4.7
853
15.8
11.8 6.9
24 5
2.4 0.5 1.8
1.4
1.4 1.9 0.6 2.4
0.8
15.4 0.7 7.5 0 0.3 2.7
mercial dried blood (Table 11), and multiplying by 100, or by multiplying directly by 18 (100 divided by 5.5). The quantity of hemoglobin may be calculated by dividing the density by 9.5 (the average in Table IV) and multiplying by 100, or by multiplying directly by 10.5 (100 divided by 9.5). With commercial dried blood, 0.10 gram should be used, with corresponding changes in the calculation. Extraction with ether is not necessary. When the color developed from meat scra s or tankage is too deep, the work may be repeated with a amaEer quantity of sample.
Dried Blood in Meat Scraps and Tankage Samples of meat scraps, tankage, and some other feeds were analyzed by the method described, with the results given in Table V. The seven samples of tankage contained from 11.9 to 31.0 per cent of dried blood. Most of the thirteen samples of meat and bone scraps were low in blood, but two contained 12.8 and 26.5 per cent, respectively.
A method for determination of the approximate quantity of blood in dried meat by-products is based upon the formation of color from hemoglobin. The sample is dissolved in sodium hydroxide, pyridine is added, color is developed by reduction with sodium hydrosulfite, and the difference in the color density with and without reduction is read a t 550 mp in a spectrophotometer. The intensity of the color is in proportion to the concentration. The maximum color is a t 562 mp with fresh blood and 550 mp with commercial dried blood, showing a change due to heating. Hemoglobin is partially destroyed by autoclaving blood, so that commercial dried blood (though part has been drained off before drying) averages 58 per cent hemoglobin compared with 73.6 per cent for fresh cow blood on a dry basis. The results of the analysis can be expressed either as dried blood or as hemoglobin. Commercial tankage (seven samples) contained 11.4 to 41.9 per cent of dried blood. Three samples of meat and bone scraps contained 6.3, 12.8, and 26.5 per cent of dried blood, while the other ten contained less than 4.1 per cent. The results are probably low.
Literature Cited (1) Assoc. American Feed Control Officials, Inc., Official Publication, 1942. (2) Drabkin, D . L.,and Austin, J. R., J. Bid. Chem., 112, 89-103 (1935). (3) Kraybill, H. R., Department of Scientific Research, American Meat Institute, private communication (1942). (4) Kraybill, H. R.. J. Poultry Sn'.. 8, 11 (1928). (5) Newcomer, H.S.,J . Bid. Chem., 37, 465-96 (1919). PRESENTED before the Divieion of Agricultural and Food Chemistry st the 103rd Meeting of the AYERICANCXZYICAL SOCIETY,Memphis, Tenn.
Analysis of Ethylene in Presence of Butane JARIES J. EBERL, University of Delaware, Newark, Del.
T
m
HE analysis of cracked gas samples containing the un-
saturated hydrocarbons ethylene, propylene, and butylene by specific absorption in sulfuric acid reagents of various concentrations in an Orsat apparatus, was described by Hurd and Spence (2). Fuming sulfuric acid was used in the ethylene pipet, and the authors noted that some absorption of butane occurred. I n spite of the nonspecific absorption of ethylene in the presence of butane, the reagent has been widely used. The disadvantages of the fuming sulfuric acid and the bromine method for the analysis of ethylene have been discussed by Gooderhani (I), who suggests the use of concentrated sulfuric acid containing 1 per cent silver sulfate as a catalyst for the analysis of ethylene in fuel gas. It was found that the reagent described by Gooderham gave rapid absorption of ethylene but was not entirely specific in the presence of butane. About 0.5 cc. of butane was absorbed per passage of 100 cc. of pure butane gas a t atmospheric pressure. I n order to eliminate the error due to butane absorption a new reagent was developed. This reagent is prepared by adding 12 grams of silver sulfate to 200 cc. of 72 per cent sulfuric acid. The reagent absorbs ethylene rapidly and gives no error due to butane absorption.
TABLEI. ABSORPTIONDATA Passage
Etbylene-n-Butane Mixture Buret Absorption
cc
cc.
I
100.0
2i:o
80.0 72.2 70.0 69.9 69.9
7.8 2.2 0.1 0.0
Total
Pure n-Butane Buret Absorption Cc.
Cc.
94.7 94.7 94.7 94.7
...
0.0 0.0 0.0 0.0
94.7 94.7
80.1
Total
0.0 0.0
A typical set of absorption data for a synthetic mixture containing 30 per cent pure ethylene and 70 per cent pure nbutane is given in Table I, with data for the passage of pure n-butane. A 2-minute time of passage was used throughout the analysis. Literature Cited (1) Gooderham, W. J., J . SOC.Chem. Ind. 57, 388T (938). (2) Hurd, C. D . , and Spence, L. U. J Am. Chem. Sac., 51, 3353 (1929).
