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Preparation of a Standard for Citric Acid Analysis
TECHNICAL NOTES
pletely under the same test conditions. These trials were both repeated using two pipes showing discoloration. Results similar to the above were obtained. The sodium hypochlorite concentration is the same in both detergent formulations. Since the quantity of detergent "B" used was about one-half that of "A," it follows that the actual level of chlorine varied in the detergent solutions. It is also important to note that efficient film removal was observed at the lower concentration of chlorine. I t has been suggested that the low metasilicate content in detergent "B" might permit greater detergent action, owing to a more favorable p H for chlorine activity (1). A cleaning procedure is described whereby an iridescent fihn was built up in a stainless steel pipeline by using a selected commercially available nonionic detergent at 120 ° F. Increasing the temperature of the nonionic and anionic cleaner to 160 ° F. did not remove the film after it had formed. A highly alkaline bottle-washing compound and an organic acid cleaner failed to remove the fihn. Chlorinated alkali detergents
PREPARATION
varied considerably in their ability to remove the iridescent fihn. O. W. K~UF~rANN Department of Microbiology and Public Health Michigan State University, East Lansing AND
P. H. TIcAc¥ Department of Food Technology University of Illinois, Urbana REFERENCES B. Personal communication. I~lenzade Products, Inc., Beloit, Wisconsin.
(1) BARRETT, R. (2) •AUFMANN,
O. W., ANDREWS, ~:~. ~I., AND
TRACY, P. ]:t. Further Studies on In-Place Cleaning. J. Dairy Sci., 38: 371. 1955. ADDENDU~J[
Since the completion of this study, a strong iridescent discoloration was observed on the glassware used in our laboratories. Replacing the commercial detergent routinely used with chlorinated alkali " B " removed the rainbow film on all test tubes and Petri plates.
OF A STANDARD FOR CITRIC ACID ANALYSIS
Considerable difficulty has been experienced with crystalline citric acid as a basis for preparation of a standard solution. Both the anhydrous and monohydrate forms of the acid are unstable at room temperature and humidity, with the result that the water content can vary between 0 and 2.5 M/M. Although the anhydrous acid is stable in a desiccator, it is difficult to obtain it by drying the monohydrate, because crystals of citric acid tend to melt at temperatures above 75 ° C. (4). Consequently, we have studied trisodium citrate as a standard material. The composition of trisodium citrate allhydrate was verified by analyzing two lots (reagent grade) obtained from different manufacturers. The salt was of high purity and was probably obtMned by reerystallization under controlled conditions of temperature and pressure (2). Sample A was taken from a freshly opened bottle, whereas the bottle containing Sample B had been opened for at least 1 yr. No preliminary drying was given either sample before analysis. Carbon was determined by dry combustion (1), water was estimated by heating to 150 ° C. overnight (4), and sodium was measured by flame photometry (5). The following results were obtained:
Found, % by wt.:
1885
CARBON (Theor. : 24.50) A B 24.28 24.36
All of these results fell within the experimental error of the various techniques. Although this study indicated that trisodium citrate dihydrate was stable at room temperature and humidity, its stability was further studied by storing for three-day intervals under various extreme conditions. Exposure to dry 80 ° C. air caused a small loss in weight (0.041%). At room temperature, storage in the desiccator resulted in a negligible loss of weight (0.003%), whereas holding at 100% relative humidity caused a gain in weight of 3.65%. I t is, therefore, apparent that trisodium citrate dihydrate is quite stable under normal laboratory conditions. I n a previous publication (3), a stock solution containing 50 mg. of anhydrous citric acid per milliliter was recommended, from which suitable standards could be prepared. On the basis of the above study of sodium citrate, the following preparation is now recommended: 76.4712 g. of trisodium citrate dihydrate (reagent grade) is made up to one liter with water. This solution is 0.260 M and, therefore, compares with one containing 50 mg. of anhydrous citric acid per milliliter. The solution is stored at 0 ° C. Fresh dilutions WATER (Theor.: 12.25) A B 12.36 12.19
SODIUM (Theor.: 23.46) A B 22.70 22.95
]886
JOURNAL OF DAIRY SCIENCE
of this stock solution are prepared each month and are also stored at 0 ° C.
J. R. MARIER M. B OULF~ Division of Applied Biology, National Research Council, Ottawa, Canada REFERENCES (1) CHRISTI~IAN,D. R., DAY, N. E., HANSELL, P. R., AND ANDERSON, R. C. Improvements in
Isotopic Carbon Assay and Chemical An-
(2) (3)
(4) (5)
alysis of Organic Compounds by Dry Combustion. Anat. Chem., 27:1935. 1955. HOLT(~N, H. H. Sodium Citrate Dihydrate. Chem. Abstrs., 33:6881. 1939. MA~mR, J. R., AND BOULET, M. Direct Determination of Citric Acid in Milk, with an Improved Pyridine---Aeetic Anhydride Method. J. Dairy Sei., 41: 1683. 1958. Merct~ Index. 6th ed. pp. 252 and 877. Merck and Co., Rahway, New Jersey. 1952. P u m ~ s , M., ANDNESSI~, N. E. Comparison Between Flame Photometric and Chemical Methods for Sodium and Potassium in Soils, Plants, Serum, and Water. Analyst, 82: 467. 1957.
T H E M E T A B O L I S M O F S E R I N E - 3 - C ~4 B Y T H E L A C T A T I N G S H E E ' P M A M M A R Y G L A N D 1.2 The contribution of serine to the metabolism of the mammary gland in capacities other than protein synthesis is unknown. Recent reports of Wood et al. (6, 7) qualitatively show that serine can be formed in the mammary gland from glucose and glycerol. The conversion most likely occurs by way of phosphoglycerate and phosphohydroxypyruvate (3). A reversal of this reaction sequence could lead to the formation of phosphoglyceraldehyde from serine. Since triose phosphate isomerase is active in the gland (7), it would be possible for serine to contribute to the formation of glycerol. To study the metabolism of serine by the mammary gland, 90 t~c. of serine-3-C 1. was placed, immediately after milking, into the gland cisterns of the udder of each of two lactating ewes. After 8 hr., the ewes were milked, sacrificed, and the glands removed for analysis. Various substances (Table 1) were isolated from the milk and lnanmmry gland tissue by conventional means and assayed for radioactivity in a thin-window gas-flow G.M. counter. Both milk and mammary tissue were analyzed to eliminate the effect of differences in secretion rates of the various nfilk components on the distribution of C~. During the 8-hr. period between the addition of the labeled compound to the udder and the sacrifice of the animal, 94 and 87% of the labeled serine were utilized from the gland cisterns in Experiments A and B, respectively. Of the serine taken up, 16% was recovered from milk and glandular tissue in Experiment A, whereas 36% was recovered from these sources in Experiment B. Of this recovered C~', 98% (Experiment A) and 99% (Experiment B) were located in the proteins of the milk and mammary gland. Table 2 shows the Authorized for publication on J'une 5, 1959, as Paper No. 2368 in the Journal Series of the Pennsylvania Agricultural Experiment Station. 2 Supported by the U. S. Public Health Service (H3632).
TABLE 1 Distribution of recovered C~* from serine-3-C 1. metabolized following administration to the lactating sheep udder" Metabolites isolated
Per cent of metabolized, recovered C1~
Milk Triglyceride glycerol Triglyceride fatty acids Phospholipids Proteins Lactose Mammary gland Triglycerlde glycerol Triglyceride fatty acids Phospholipids Proteins Glycogen
Exp. A Trace 0.0 0.2 33.6 0.0
Exp. B Trace 0.0 0.1 82.9 0.2
0.4 0.0 0.9 64.7 Trace
Trace 0.0 0.1 16.7 Trace
In Experiment A, 94% of the administered seriae was metabolized, of which 16% was recovered in the substances isolated. In Experiment B, 87% of the serine was metabolized, of which 36(/c was recovered. TABLE 2 Specific activity of the classical fractions of milk proteins following the arministratlon of serine-3-C ~4 to the lactating sheep udder (Experiment A) Protein fraction a Casein Lactoglobulin Lactalbumin
Specific activity (c.p.m/mg) 147 49 103
Nomenclature as suggested by Reference 4. specific activity of the classical protein fractions in the milk from Experiment A. The specific activity of casein and lactalburain, in relation to the per cent serine in each (casein 6.3, lactalbumin 4.8), suggests that
pletely under the same test conditions. These trials were both repeated using two pipes showing discoloration. Results similar to the above were obtained. The sodium hypochlorite concentration is the same in both detergent formulations. Since the quantity of detergent "B" used was about one-half that of "A," it follows that the actual level of chlorine varied in the detergent solutions. It is also important to note that efficient film removal was observed at the lower concentration of chlorine. I t has been suggested that the low metasilicate content in detergent "B" might permit greater detergent action, owing to a more favorable p H for chlorine activity (1). A cleaning procedure is described whereby an iridescent fihn was built up in a stainless steel pipeline by using a selected commercially available nonionic detergent at 120 ° F. Increasing the temperature of the nonionic and anionic cleaner to 160 ° F. did not remove the film after it had formed. A highly alkaline bottle-washing compound and an organic acid cleaner failed to remove the fihn. Chlorinated alkali detergents
PREPARATION
varied considerably in their ability to remove the iridescent fihn. O. W. K~UF~rANN Department of Microbiology and Public Health Michigan State University, East Lansing AND
P. H. TIcAc¥ Department of Food Technology University of Illinois, Urbana REFERENCES B. Personal communication. I~lenzade Products, Inc., Beloit, Wisconsin.
(1) BARRETT, R. (2) •AUFMANN,
O. W., ANDREWS, ~:~. ~I., AND
TRACY, P. ]:t. Further Studies on In-Place Cleaning. J. Dairy Sci., 38: 371. 1955. ADDENDU~J[
Since the completion of this study, a strong iridescent discoloration was observed on the glassware used in our laboratories. Replacing the commercial detergent routinely used with chlorinated alkali " B " removed the rainbow film on all test tubes and Petri plates.
OF A STANDARD FOR CITRIC ACID ANALYSIS
Considerable difficulty has been experienced with crystalline citric acid as a basis for preparation of a standard solution. Both the anhydrous and monohydrate forms of the acid are unstable at room temperature and humidity, with the result that the water content can vary between 0 and 2.5 M/M. Although the anhydrous acid is stable in a desiccator, it is difficult to obtain it by drying the monohydrate, because crystals of citric acid tend to melt at temperatures above 75 ° C. (4). Consequently, we have studied trisodium citrate as a standard material. The composition of trisodium citrate allhydrate was verified by analyzing two lots (reagent grade) obtained from different manufacturers. The salt was of high purity and was probably obtMned by reerystallization under controlled conditions of temperature and pressure (2). Sample A was taken from a freshly opened bottle, whereas the bottle containing Sample B had been opened for at least 1 yr. No preliminary drying was given either sample before analysis. Carbon was determined by dry combustion (1), water was estimated by heating to 150 ° C. overnight (4), and sodium was measured by flame photometry (5). The following results were obtained:
Found, % by wt.:
1885
CARBON (Theor. : 24.50) A B 24.28 24.36
All of these results fell within the experimental error of the various techniques. Although this study indicated that trisodium citrate dihydrate was stable at room temperature and humidity, its stability was further studied by storing for three-day intervals under various extreme conditions. Exposure to dry 80 ° C. air caused a small loss in weight (0.041%). At room temperature, storage in the desiccator resulted in a negligible loss of weight (0.003%), whereas holding at 100% relative humidity caused a gain in weight of 3.65%. I t is, therefore, apparent that trisodium citrate dihydrate is quite stable under normal laboratory conditions. I n a previous publication (3), a stock solution containing 50 mg. of anhydrous citric acid per milliliter was recommended, from which suitable standards could be prepared. On the basis of the above study of sodium citrate, the following preparation is now recommended: 76.4712 g. of trisodium citrate dihydrate (reagent grade) is made up to one liter with water. This solution is 0.260 M and, therefore, compares with one containing 50 mg. of anhydrous citric acid per milliliter. The solution is stored at 0 ° C. Fresh dilutions WATER (Theor.: 12.25) A B 12.36 12.19
SODIUM (Theor.: 23.46) A B 22.70 22.95
]886
JOURNAL OF DAIRY SCIENCE
of this stock solution are prepared each month and are also stored at 0 ° C.
J. R. MARIER M. B OULF~ Division of Applied Biology, National Research Council, Ottawa, Canada REFERENCES (1) CHRISTI~IAN,D. R., DAY, N. E., HANSELL, P. R., AND ANDERSON, R. C. Improvements in
Isotopic Carbon Assay and Chemical An-
(2) (3)
(4) (5)
alysis of Organic Compounds by Dry Combustion. Anat. Chem., 27:1935. 1955. HOLT(~N, H. H. Sodium Citrate Dihydrate. Chem. Abstrs., 33:6881. 1939. MA~mR, J. R., AND BOULET, M. Direct Determination of Citric Acid in Milk, with an Improved Pyridine---Aeetic Anhydride Method. J. Dairy Sei., 41: 1683. 1958. Merct~ Index. 6th ed. pp. 252 and 877. Merck and Co., Rahway, New Jersey. 1952. P u m ~ s , M., ANDNESSI~, N. E. Comparison Between Flame Photometric and Chemical Methods for Sodium and Potassium in Soils, Plants, Serum, and Water. Analyst, 82: 467. 1957.
T H E M E T A B O L I S M O F S E R I N E - 3 - C ~4 B Y T H E L A C T A T I N G S H E E ' P M A M M A R Y G L A N D 1.2 The contribution of serine to the metabolism of the mammary gland in capacities other than protein synthesis is unknown. Recent reports of Wood et al. (6, 7) qualitatively show that serine can be formed in the mammary gland from glucose and glycerol. The conversion most likely occurs by way of phosphoglycerate and phosphohydroxypyruvate (3). A reversal of this reaction sequence could lead to the formation of phosphoglyceraldehyde from serine. Since triose phosphate isomerase is active in the gland (7), it would be possible for serine to contribute to the formation of glycerol. To study the metabolism of serine by the mammary gland, 90 t~c. of serine-3-C 1. was placed, immediately after milking, into the gland cisterns of the udder of each of two lactating ewes. After 8 hr., the ewes were milked, sacrificed, and the glands removed for analysis. Various substances (Table 1) were isolated from the milk and lnanmmry gland tissue by conventional means and assayed for radioactivity in a thin-window gas-flow G.M. counter. Both milk and mammary tissue were analyzed to eliminate the effect of differences in secretion rates of the various nfilk components on the distribution of C~. During the 8-hr. period between the addition of the labeled compound to the udder and the sacrifice of the animal, 94 and 87% of the labeled serine were utilized from the gland cisterns in Experiments A and B, respectively. Of the serine taken up, 16% was recovered from milk and glandular tissue in Experiment A, whereas 36% was recovered from these sources in Experiment B. Of this recovered C~', 98% (Experiment A) and 99% (Experiment B) were located in the proteins of the milk and mammary gland. Table 2 shows the Authorized for publication on J'une 5, 1959, as Paper No. 2368 in the Journal Series of the Pennsylvania Agricultural Experiment Station. 2 Supported by the U. S. Public Health Service (H3632).
TABLE 1 Distribution of recovered C~* from serine-3-C 1. metabolized following administration to the lactating sheep udder" Metabolites isolated
Per cent of metabolized, recovered C1~
Milk Triglyceride glycerol Triglyceride fatty acids Phospholipids Proteins Lactose Mammary gland Triglycerlde glycerol Triglyceride fatty acids Phospholipids Proteins Glycogen
Exp. A Trace 0.0 0.2 33.6 0.0
Exp. B Trace 0.0 0.1 82.9 0.2
0.4 0.0 0.9 64.7 Trace
Trace 0.0 0.1 16.7 Trace
In Experiment A, 94% of the administered seriae was metabolized, of which 16% was recovered in the substances isolated. In Experiment B, 87% of the serine was metabolized, of which 36(/c was recovered. TABLE 2 Specific activity of the classical fractions of milk proteins following the arministratlon of serine-3-C ~4 to the lactating sheep udder (Experiment A) Protein fraction a Casein Lactoglobulin Lactalbumin
Specific activity (c.p.m/mg) 147 49 103
Nomenclature as suggested by Reference 4. specific activity of the classical protein fractions in the milk from Experiment A. The specific activity of casein and lactalburain, in relation to the per cent serine in each (casein 6.3, lactalbumin 4.8), suggests that