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Ashing of Egg Whites and Yolks and the Determination of Their Iron Content*
Ashing of Egg Whites and Yolks and the Determination of Their Iron Content* SELMA L. BANDEMER, J. A. DAVIDSON, AND P. J. SCHAIBLE
Chemistry Section and Poultry Section, Michigan Agricultural Experiment Station (Received for publication March 7, 1944)
ONSIDERABLE difficulty was experienced in ashing the whites and yolks of individual eggs preliminary to the determination of their iron content. This was especially true with the whites where it was necessary to use the entire amount of each egg in order to obtain sufficient iron for analysis. Ashing this quantity in the muffle furnace was impossible as the material foamed out of the crucibles. Drying the egg whites did not prevent the foaming. The following experiments were conducted, therefore, to overcome this difficulty and, at the same time, obtain an acidsoluble ash from which the iron could be easily and completely recovered.
were folded in quarters and placed within No. 3 crucibles. Egg white was introduced within the filter paper and ashed over the Meeker burner and in the muffle furnace as before. The filter paper kept the egg white away from the crucible walls and thus aided the escape of the moisture and gas. It also tended to reduce fusing but did not overcome it entirely. A muffle furnace temperature low enough to avoid fusing was insufficient to burn off the carbon. The high-phosphorus concentration in relation to the base in egg white suggested the possibility that additions of magnesium chloride might be beneficial during ashing. To determine whether or not it was effective, the following experiments were carried out.
EXPERIMENTAL
Egg White—After much trial and error, it was found that the maximum amount of egg white from individual eggs (38 ml.) could be kept within the confines of No. 3 (75 ml.) crucibles if the egg whites were heated rapidly over Meeker burners until the moisture and gas were driven off and a light porous char formed. The ashing was then completed in the muffle furnace overnight. Nevertheless, under these conditions, the ash fused somewhat—even during the preliminary charring. In an attempt to prevent local overheating and in order to aid the release of the moisture and gas, the following procedure was devised. Filter papers * Journal Article No. 691 (n.s.) Michigan Agricultural Experiment Station.
A solution of ferrous sulfate was mixed with solutions of various salts of phosphoric acid so that the Fe to P ratio was 1:100, as in egg white. Two series of aliquots of these mixed solutions were evaporated to dryness on the steam bath; one series was treated with l.S ml. of 33 percent solution of magnesium chloride (MgCl 2 -6H 2 0) whereas the other served as controls. All samples were then heated overnight in the muffle furnace at 600°C, cooled, taken up in 15 ml. of 1 ;4 hydrochloric acid by warming on an electric hot-plate, made up to 25 ml. and analyzed for iron by the o-phenanthroline method as modified by the authors (1944). The untreated samples fused somewhat and dissolved incompletely and difficultly in dilute hydrochloric acid. Consequently, recoveries were poor, par-
437
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
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438
SELMA L. BANDEMER, J. A. DAVIDSON, AND P. J. SCHAIBLE
ticularly with the dibasic phosphates (Table 1). On the other hand, excellent recoveries were obtained when the samples were treated with magnesium chloride. In the case of the ammonium phosphates, the results were slightly low but more magTABLE 1.—Jifiect of addition of magnesium chloride upon the recovery of iron from an ash of a ferrous sulfate-phosphate salt mixture ( F e : P = 1:100) Percent recovery Treated Untreated w i t h M g C l 2
None KH 2 P0 4 K2HPO4 NaHkPCVEkO NaOIPCV 12H 2 0 Na 3 P0 4 - 12H 2 0 (NH4)H2P04 (NH 4 )H 2 P0 4 * (NH^jHPO* (NH4)2HP04* CaH 4 (P0 4 ) 2 H 2 0 CaHP0 4 -2H 2 0 N a ^ O r - 10H2O NaNH4HP(V4H 2 0 H a P0 4
100 97 88 80 74 95 85 82 82 88 92 98 80 69 67
100 100 100 100 1Q0 100 94 100 98 100 100 100 100 98 100
* Increased amount of MgCl 2 .
nesium chloride corrected this. The magnesium salt prevented fusing in the muffle and produced a light fluffy ash which dissolved easily and completely in dilute hydrochloric acid. A second experiment was made to learn the effect of magnesium chloride upon the recovery of iron from egg white (Table 2). If magnesium chloride was not used during ashing, the values for egg white were
The procedure as finally developed for egg whites and yolks follows. DETERMINATION OF IRON IN EGG WHITES AND YOLKS
(a) Magnesium chloride solution.—Dissolve 50 grams MgCl 2 -6H 2 0 and dilute to 150 ml. (b) 1:4 Hydrochloric acid. (c) Sodium citrate solution.—Dissolve 250 grams of C.P. sodium citrate in dis-
TABLE 2.—Recovery of iron added to egg-white char, untreated and treated with magnesium chloride. Iron in egg white expressed in micrograms per 100 grams Treatment with MgCls
Untreated Treated Treated Treated
Iron
Samples of egg white A
B
C
121 138 230 327
80 137 234 334
84 137 229 336
D • 91 140 234 • 329
Average
Added
Recovered
micrograms 0 0 95 200
micrograms
94 138 232 331
— —
94 193
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
Phosphate salt
low and erratic. On the other hand, if it was present, the iron values were higher and consistent. When iron was added to the whites whose chars were subsequently treated with magnesium chloride, very good recoveries were obtained. Egg Yolk—Inasmuch as egg yolk contains a rather high concentration of iron, only a small amount of yolk was needed for analysis and, consequently, no difficulties were encountered. In some experiments in which it was desired to ash larger quantities of yolk, the previously described filter-paper technique was used efficaciously to take care of the large amount of fat. Indeed, this technic probably could be applied with advantage in ashing largesized samples of proteinaceous and fatty materials which are difficult to handle by the usual methods. That difficulties are encountered in ashing materials for iron determination is evidenced by the Report of Methods Committee on the determination of iron in cereal products (Sullivan, 1943).
DETERMINATION OF IRON CONTENT OF EGG WHITES AND YOLKS
PROCEDURE
Each egg was broken into a tared dish and weighed. Eggs with blood spots were discarded; meat spots were removed. The white was pipetted into a crucible prepared as follows: a 12.5 cm. No. 40 Whatman filter paper was folded as usual for filtering and the bottom of the cone folded back about one cm. The paper was fitted in a No, 3 crucible which was then weighed with a tared cover glass. After the white had been weighed in the covered crucible, it was burned off over a Meeker burner to a porous char. This char was moistened with 1.5 ml. magnesium chloride solution, added dropwise, and then ashed at 600°C. overnight in a muffle furnace. After the white had been removed, the yolk was placed upon a filter paper and rolled back and forth to absorb the film of egg white on the vitelline membrane. It was weighed and the vitelline membrane pricked with a glass teaser, the yolk being allowed to run into a 30 ml. beaker. After mixing, a sample of 3 to 4 grams was transferred to a tared No. 1 crucible, weighed, and placed in the muffle at 575°C. overnight. The ash of the white was dissolved in
15 ml. of a 1:4 hydrochloric acid solution on the hot-plate; that of the yolk in 20 ml. The solutions were transferred to 25 ml. volumetric flasks and made up to volume. Aliquots of 5 ml. for yolk or 10 ml. for white were pipetted into 25 ml. volumetric flasks, 1 ml. of hydroquinone and 2 ml. of o-phenanthroline solutions added and the flasks shaken. To yolk samples, 5 ml., and to white, 4 ml. of sodium citrate solution were added and the solutions made up to volume. The quantities of citrate mentioned represent the average requirements of a number of individual titrations (Bandemer and Schaible, 1944). Samples were allowed to stand at least 0.5 hour for the development of maximum color and then read in the photometer (Cenco Sheard-Sanford Photelometer was used) in 1 cm. absorption cells using a Corning No. 430 filter of 12.5 mm. thickness. A blank solution of the reagents was employed for the 100 setting of the photometer so that the readings were automatically corrected for the amount of iron in the reagents. Iron concentrations were read off a curve made by running known amounts of the iron standard solution exactly as was done for the unknowns. SUMMARY
In attempting to ash egg whites preliminary to determining their iron content, the material was found to foam out of crucibles and the ash was found to fuse. These difficulties were overcome as follows: Egg whites were placed in folded filter papers within crucibles and charred over Meeker burners; magnesium chloride was then added and ashing completed in the muffle furnace overnight at 600°C. The difficulties encountered were attributed to the high-phosphorus content of the white relative to its content of base because when various phosphorus compounds were added to iron solutions in the
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
tilled water and make up to 1 liter. (d) Hydroquinone solution.—Dissolve 1 gram of hydroquinone in 100 ml. distilled water. Store in refrigerator and discard if any color develops. (e) o-Phenanthroline solution.—Add 0.5 grams o-phenanthroline (monohydrate) to 175 ml. hot distilled water. Stir to dissolve and, when cool, make up to 200 ml. Store in refrigerator and discard if any color develops. (f) Iron standard solution.—(1 mg. Fe per ml.) Dissolve 1 gram of electrolytic iron in 50 ml. 10 percent H 2 S0 4 solution and make up to 1 liter with distilled water.
439
440
NEWS AND NOTES
Details of the procedures for ashing individual egg whites and yolks and for determining their iron content by the o-phenanthroline method are given. REFERENCES
Bandemer, S. L. and P. J. Schaible, 1944. A study of the o-phenanthroline method for the determination of iron. Ind. Eng. Chem., Anal. Ed. 16:317-319. Sullivan, B., 1943. Report of methods committee on the determination of iron in cereal products. Cereal Chem. 20:36-38.
News and Notes In the "Notice to Membership" appearing on page 430 is an important item regarding the journal. The change in the editorship of this journal will not be made until approximately November 1. Consequently, all correspondence regarding manuscripts or other details of publication should be sent to Dr. D. R. Marble, 1503 East Main Street, Warsaw, Indiana. After November 1 such correspondence should go to Dr. R. George Jaap, Poultry Department, Oklahoma A. & M. College, Stillwater, Oklahoma. Dr. Homer Patrick (B.S. 1939; M.S. 1940) University of Missouri and Ph.D. (1942) at the Pennsylvania State College has resigned at the South Carolina Agricultural Experiment Station, Clemson, South Carolina to become manager of the Biological Laboratory, Purina Mills, St. Louis, Missouri. As a result of the resignation of Dr. D. R. Marble, Professor of Poultry Husbandry at the Pennsylvania State College to accept a position with Creighton Brothers, War-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
proportion found in egg white, the ash fused and the iron was not completely recovered. If magnesium chloride was added before the materials were placed in the muffle furnace, no fusing occurred and the iron was completely recovered. No difficulties were encountered in ashing small samples of untreated egg yolk nor in determining the iron content. For larger samples of yolk, as well as for other similar materials, the use of the folded filter paper in the crucible was found advantageous.
saw, Indiana, several other changes have been announced. The Pennsylvania State College position will be filled by Dr. A. J. G. Maw, Professor and Acting Head of the Poultry Department, Iowa State College, Ames, Iowa. Just as this issue goes to press word has been received of the appointment at the Iowa State College as Professor and Acting Head of Robert E. Phillips (B.S. 1935; M.S. 1936) Kansas State College and Ph.D. (1941) Iowa State College. Dr. Phillips was formerly Associate Professor of avian physiology at the University of Maryland. Copies of May Issue Wanted—As a result of an error in estimating the number of copies to be printed of the May, 1944 issue the usual number of reserve copies is not available. Any member or subscriber willing to release his copy will assist the editor's office greatly by sending it to D. R. Marble, 1503 East Main Street, Warsaw, Indiana. A refund of 75$ will be made on each copy returned to the editor's office.
Chemistry Section and Poultry Section, Michigan Agricultural Experiment Station (Received for publication March 7, 1944)
ONSIDERABLE difficulty was experienced in ashing the whites and yolks of individual eggs preliminary to the determination of their iron content. This was especially true with the whites where it was necessary to use the entire amount of each egg in order to obtain sufficient iron for analysis. Ashing this quantity in the muffle furnace was impossible as the material foamed out of the crucibles. Drying the egg whites did not prevent the foaming. The following experiments were conducted, therefore, to overcome this difficulty and, at the same time, obtain an acidsoluble ash from which the iron could be easily and completely recovered.
were folded in quarters and placed within No. 3 crucibles. Egg white was introduced within the filter paper and ashed over the Meeker burner and in the muffle furnace as before. The filter paper kept the egg white away from the crucible walls and thus aided the escape of the moisture and gas. It also tended to reduce fusing but did not overcome it entirely. A muffle furnace temperature low enough to avoid fusing was insufficient to burn off the carbon. The high-phosphorus concentration in relation to the base in egg white suggested the possibility that additions of magnesium chloride might be beneficial during ashing. To determine whether or not it was effective, the following experiments were carried out.
EXPERIMENTAL
Egg White—After much trial and error, it was found that the maximum amount of egg white from individual eggs (38 ml.) could be kept within the confines of No. 3 (75 ml.) crucibles if the egg whites were heated rapidly over Meeker burners until the moisture and gas were driven off and a light porous char formed. The ashing was then completed in the muffle furnace overnight. Nevertheless, under these conditions, the ash fused somewhat—even during the preliminary charring. In an attempt to prevent local overheating and in order to aid the release of the moisture and gas, the following procedure was devised. Filter papers * Journal Article No. 691 (n.s.) Michigan Agricultural Experiment Station.
A solution of ferrous sulfate was mixed with solutions of various salts of phosphoric acid so that the Fe to P ratio was 1:100, as in egg white. Two series of aliquots of these mixed solutions were evaporated to dryness on the steam bath; one series was treated with l.S ml. of 33 percent solution of magnesium chloride (MgCl 2 -6H 2 0) whereas the other served as controls. All samples were then heated overnight in the muffle furnace at 600°C, cooled, taken up in 15 ml. of 1 ;4 hydrochloric acid by warming on an electric hot-plate, made up to 25 ml. and analyzed for iron by the o-phenanthroline method as modified by the authors (1944). The untreated samples fused somewhat and dissolved incompletely and difficultly in dilute hydrochloric acid. Consequently, recoveries were poor, par-
437
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
C
438
SELMA L. BANDEMER, J. A. DAVIDSON, AND P. J. SCHAIBLE
ticularly with the dibasic phosphates (Table 1). On the other hand, excellent recoveries were obtained when the samples were treated with magnesium chloride. In the case of the ammonium phosphates, the results were slightly low but more magTABLE 1.—Jifiect of addition of magnesium chloride upon the recovery of iron from an ash of a ferrous sulfate-phosphate salt mixture ( F e : P = 1:100) Percent recovery Treated Untreated w i t h M g C l 2
None KH 2 P0 4 K2HPO4 NaHkPCVEkO NaOIPCV 12H 2 0 Na 3 P0 4 - 12H 2 0 (NH4)H2P04 (NH 4 )H 2 P0 4 * (NH^jHPO* (NH4)2HP04* CaH 4 (P0 4 ) 2 H 2 0 CaHP0 4 -2H 2 0 N a ^ O r - 10H2O NaNH4HP(V4H 2 0 H a P0 4
100 97 88 80 74 95 85 82 82 88 92 98 80 69 67
100 100 100 100 1Q0 100 94 100 98 100 100 100 100 98 100
* Increased amount of MgCl 2 .
nesium chloride corrected this. The magnesium salt prevented fusing in the muffle and produced a light fluffy ash which dissolved easily and completely in dilute hydrochloric acid. A second experiment was made to learn the effect of magnesium chloride upon the recovery of iron from egg white (Table 2). If magnesium chloride was not used during ashing, the values for egg white were
The procedure as finally developed for egg whites and yolks follows. DETERMINATION OF IRON IN EGG WHITES AND YOLKS
(a) Magnesium chloride solution.—Dissolve 50 grams MgCl 2 -6H 2 0 and dilute to 150 ml. (b) 1:4 Hydrochloric acid. (c) Sodium citrate solution.—Dissolve 250 grams of C.P. sodium citrate in dis-
TABLE 2.—Recovery of iron added to egg-white char, untreated and treated with magnesium chloride. Iron in egg white expressed in micrograms per 100 grams Treatment with MgCls
Untreated Treated Treated Treated
Iron
Samples of egg white A
B
C
121 138 230 327
80 137 234 334
84 137 229 336
D • 91 140 234 • 329
Average
Added
Recovered
micrograms 0 0 95 200
micrograms
94 138 232 331
— —
94 193
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
Phosphate salt
low and erratic. On the other hand, if it was present, the iron values were higher and consistent. When iron was added to the whites whose chars were subsequently treated with magnesium chloride, very good recoveries were obtained. Egg Yolk—Inasmuch as egg yolk contains a rather high concentration of iron, only a small amount of yolk was needed for analysis and, consequently, no difficulties were encountered. In some experiments in which it was desired to ash larger quantities of yolk, the previously described filter-paper technique was used efficaciously to take care of the large amount of fat. Indeed, this technic probably could be applied with advantage in ashing largesized samples of proteinaceous and fatty materials which are difficult to handle by the usual methods. That difficulties are encountered in ashing materials for iron determination is evidenced by the Report of Methods Committee on the determination of iron in cereal products (Sullivan, 1943).
DETERMINATION OF IRON CONTENT OF EGG WHITES AND YOLKS
PROCEDURE
Each egg was broken into a tared dish and weighed. Eggs with blood spots were discarded; meat spots were removed. The white was pipetted into a crucible prepared as follows: a 12.5 cm. No. 40 Whatman filter paper was folded as usual for filtering and the bottom of the cone folded back about one cm. The paper was fitted in a No, 3 crucible which was then weighed with a tared cover glass. After the white had been weighed in the covered crucible, it was burned off over a Meeker burner to a porous char. This char was moistened with 1.5 ml. magnesium chloride solution, added dropwise, and then ashed at 600°C. overnight in a muffle furnace. After the white had been removed, the yolk was placed upon a filter paper and rolled back and forth to absorb the film of egg white on the vitelline membrane. It was weighed and the vitelline membrane pricked with a glass teaser, the yolk being allowed to run into a 30 ml. beaker. After mixing, a sample of 3 to 4 grams was transferred to a tared No. 1 crucible, weighed, and placed in the muffle at 575°C. overnight. The ash of the white was dissolved in
15 ml. of a 1:4 hydrochloric acid solution on the hot-plate; that of the yolk in 20 ml. The solutions were transferred to 25 ml. volumetric flasks and made up to volume. Aliquots of 5 ml. for yolk or 10 ml. for white were pipetted into 25 ml. volumetric flasks, 1 ml. of hydroquinone and 2 ml. of o-phenanthroline solutions added and the flasks shaken. To yolk samples, 5 ml., and to white, 4 ml. of sodium citrate solution were added and the solutions made up to volume. The quantities of citrate mentioned represent the average requirements of a number of individual titrations (Bandemer and Schaible, 1944). Samples were allowed to stand at least 0.5 hour for the development of maximum color and then read in the photometer (Cenco Sheard-Sanford Photelometer was used) in 1 cm. absorption cells using a Corning No. 430 filter of 12.5 mm. thickness. A blank solution of the reagents was employed for the 100 setting of the photometer so that the readings were automatically corrected for the amount of iron in the reagents. Iron concentrations were read off a curve made by running known amounts of the iron standard solution exactly as was done for the unknowns. SUMMARY
In attempting to ash egg whites preliminary to determining their iron content, the material was found to foam out of crucibles and the ash was found to fuse. These difficulties were overcome as follows: Egg whites were placed in folded filter papers within crucibles and charred over Meeker burners; magnesium chloride was then added and ashing completed in the muffle furnace overnight at 600°C. The difficulties encountered were attributed to the high-phosphorus content of the white relative to its content of base because when various phosphorus compounds were added to iron solutions in the
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
tilled water and make up to 1 liter. (d) Hydroquinone solution.—Dissolve 1 gram of hydroquinone in 100 ml. distilled water. Store in refrigerator and discard if any color develops. (e) o-Phenanthroline solution.—Add 0.5 grams o-phenanthroline (monohydrate) to 175 ml. hot distilled water. Stir to dissolve and, when cool, make up to 200 ml. Store in refrigerator and discard if any color develops. (f) Iron standard solution.—(1 mg. Fe per ml.) Dissolve 1 gram of electrolytic iron in 50 ml. 10 percent H 2 S0 4 solution and make up to 1 liter with distilled water.
439
440
NEWS AND NOTES
Details of the procedures for ashing individual egg whites and yolks and for determining their iron content by the o-phenanthroline method are given. REFERENCES
Bandemer, S. L. and P. J. Schaible, 1944. A study of the o-phenanthroline method for the determination of iron. Ind. Eng. Chem., Anal. Ed. 16:317-319. Sullivan, B., 1943. Report of methods committee on the determination of iron in cereal products. Cereal Chem. 20:36-38.
News and Notes In the "Notice to Membership" appearing on page 430 is an important item regarding the journal. The change in the editorship of this journal will not be made until approximately November 1. Consequently, all correspondence regarding manuscripts or other details of publication should be sent to Dr. D. R. Marble, 1503 East Main Street, Warsaw, Indiana. After November 1 such correspondence should go to Dr. R. George Jaap, Poultry Department, Oklahoma A. & M. College, Stillwater, Oklahoma. Dr. Homer Patrick (B.S. 1939; M.S. 1940) University of Missouri and Ph.D. (1942) at the Pennsylvania State College has resigned at the South Carolina Agricultural Experiment Station, Clemson, South Carolina to become manager of the Biological Laboratory, Purina Mills, St. Louis, Missouri. As a result of the resignation of Dr. D. R. Marble, Professor of Poultry Husbandry at the Pennsylvania State College to accept a position with Creighton Brothers, War-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 15, 2015
proportion found in egg white, the ash fused and the iron was not completely recovered. If magnesium chloride was added before the materials were placed in the muffle furnace, no fusing occurred and the iron was completely recovered. No difficulties were encountered in ashing small samples of untreated egg yolk nor in determining the iron content. For larger samples of yolk, as well as for other similar materials, the use of the folded filter paper in the crucible was found advantageous.
saw, Indiana, several other changes have been announced. The Pennsylvania State College position will be filled by Dr. A. J. G. Maw, Professor and Acting Head of the Poultry Department, Iowa State College, Ames, Iowa. Just as this issue goes to press word has been received of the appointment at the Iowa State College as Professor and Acting Head of Robert E. Phillips (B.S. 1935; M.S. 1936) Kansas State College and Ph.D. (1941) Iowa State College. Dr. Phillips was formerly Associate Professor of avian physiology at the University of Maryland. Copies of May Issue Wanted—As a result of an error in estimating the number of copies to be printed of the May, 1944 issue the usual number of reserve copies is not available. Any member or subscriber willing to release his copy will assist the editor's office greatly by sending it to D. R. Marble, 1503 East Main Street, Warsaw, Indiana. A refund of 75$ will be made on each copy returned to the editor's office.