V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3 However, a t higher stirring velocities and with the propeller stirrer the relations are much more intricate. The maxima of the curves for 11 and 12y0 pastes in Figure 1 are not proportionally higher than that of the 10% paste. The viscosity of the cooking paste (heated for 18 minutes and more) is the same for the three concentrations, Evidently a compensation occurs: The breakdown of the granules a t higher concentrations is so much more rapid that the higher total volume of the particles is counterbalanced. In all probability the rate of breakdown is related to the amount of liquid between the particles. When this decreases below a certain minimum value necessary for a free movement of the swollen grains, the stirring energy will be used for deformation and shear of the particles themselves to a greater extent than in the case of free moving particles. The impression is obtained that this occurs in many samples a t concentrations of 10% and more. Differences between the samples have a tendency to diminish under these circumstances.
311
If in comparing different samples one is interested in the comequences of the water-absorption capacitv of the starch, the concentration used must be so much lower that the majority of the samples reach the maximal swelling stage. h concentration of 9% is recommended for this purpose. A moderate rate of stirring, provided its action is sufficient to produce a paste free from lumps, gives a better opportunity to observe the swelling stage. LITERATURE CITED
-4nker, C. A . , and Geddes, W.F., Cereal Chern., 21,335 (19441. Bechtel. W. G., and Fischer, E. K.. J . Colload Sci., 4, 265 (1949). Farrow, F. D., Lowe, G. M., and Seale, S. XI., J . Teztile Inst., 19, T 18 (1928).
Fischer, E, K., and Llndsley, C. H., J . Colloid Sci., 3, 111 (1948). Hofstee, J., Chem. Weekblad, 46, 515 (1950). Kesler, C. C., and Bechtel, W. G , A N ~ L .CHEM.,19, 16 (1947). Selling, H. J., and Lamoen, F 1,. .J van, Cht-n~.Weekblad, 43, 602 (1 947). RECEIVED for review M a y 16, 1952. Accepted October 22. 1952.
Gravimetric Estimation of Proteins Precipitated by Trichloroacetic Acid FREDERIC L. HOCH AND BERT L. VALLEE Department of Biology, Massachusetts Institute of Technology, Cambridge, >Mass. Gravimetric measurement has been the primary method for the quantitation of proteins. Trichloroacetic acid has been used widely as a specific and quantitative precipitating agent for proteins and to separate them from nonprotein substances, proteinhound cations, and organic groups. However, no attention has been devoted to the quantitation of the proteins thus precipitated. Weighing of these protein residues, properly dried, may allow estimation of anhydrous mass and direct correlation with properties of these proteins while in solution. Pure human and bovine serum albumin, pure bovine insulin, and crude bovine pituitary extract were analyzed by weighing dried precipitates after treatment with trichloroacetic acid. Such weights are in excellent agreement with weights of crystalline proteins, with protein determinations by optical
G
RAVIMETRIC measurement has been the primary method for the determination of protein:; other methods have been based upon this primary referent (6, 18, 24). Proteins in solution have been so estimated either directly, after drying (6, go), or after precipitation by a variety of agents with removal of nonprotein materials by washing and subsequent drying of the precipitate (3,4, 7 , f Q ) . Reproducible results have been obtained by drying of proteins to constant weight a t temperatures between 80' and 11.0" C. (6,20). Trichloroacetic acid has been used routinely in many laboratories a? a specific and quantitative precipitating agent for proteins. There is no report of a critical appraisal of such a method for the gravimetric measurement of proteins. The method described utilizes the specificity of trichloroacetic acid in the precipitation of proteins, allows direct measurement of weight of dried proteins, is applicable to solutions or precipitates of proteins, and can be used profitably to measure amounts of proteins whenever trichloroacetic acid precipitation is being
measurements at 280 m,u, and estimates of weight on the basis of nitrogen determinations. A coefficient of variation of +3.8% was attained for 5-mg. amounts of protein. The method results in quantitative recovery of proteins from solution. Proteins are not hydrolyzed. Molecular weight does not appear to be the determining factor in trichloroacetic precipitation of proteins of molecular weight above 12,000. This procedure allows measurement of dried protein residues quantitatively recovered from solution. It permits studies of the presumable hydration of proteins and frees them from associated organic or inorganic molecules, which can be determined separately in the supernatant. It is possible to relate amounts of cations and other molecules dissociated by the precipitating agent from the proteins to their absolute protein dry weight.
employed for other purposes. Drying was performed a t a temperature of 110' C. which is sufficient to eliminate water by evaporation and trichloroacetic acid by decomposition. i\ number of purified proteins and mixtures of proteine with other substances were estimated following precipitation with trichloroacetic acid; this method has been applied in other studies (14, f 6 , 87). The validity of this procedure is here appraised by comparison of weights of dried precipitated proteins with weights obtained by direct weighing of pure dry proteins, spectre photometric measurements on clear solutions of dissolved proteins. and nitrogen determinations. METHOD
The weights of the following purified proteins were estimated: crystalline bovine serum albumin, human serum albumin (Cutter Laboratories), and electrodialyzed amorphous bovine insulin (prepared by D. F. Waugh). A partially purified pituitary extract (Armour) was also used. All proteins and proteincontaining preparations were dialyzed against large volumes of
318
ANALYTICAL CHEMISTRY
distilled water for a t least 24 hours a t 4" C. to remove nonprotein materials before further procedures were applied. All weights were determined on an analytical balance (Seederer-Kohlbusch) having a precision of f 0 . 2 mg. Fresh calcium chloride was placed in the balance chamber as a drying agent. Marked borosilicate glass 15-ml. centrifuge tubes were weighed in a small weighed beaker; each tube and the beaker were weighed empty after drying for a t least 3 hours a t 110' C. All protein samples were dried at 110' C. in n-eighed centrifuge tubes. A drying oven was used a t atmospheric pressure, and dried materials were cooled before weighing by allowing them t o stand exposed to room atmosphere. Some samples of crystalline bovine serum albumin were dried in a vacuum oven; some were cooled in a desiccator over calcium chloride. One- t o 2-ml. aliquots of protein solutions or suspensions in centrifuge tubes were either dried immediately or 2 ml. of 20y0 trichloroacetic acid were added, mixed with a fin'e borosilicate glass rod, and allowed t o stand either a t room temperature or a t 90' C. for 15 minutes in a heated water bath, with occasional stirring. After centrifugation a t 3000 r.p.m. for 15 minutes, the supernatant fluid was decanted. The precipitate and centrifuge tube were dried until minimum weight was achieved, as indicated by weighings a t 24-hour intervals. The amounts of protein in the dialyzed preparations used were also determined by other methods. Nitrogen was measured by a micro-Kjeldahl method by B. A. Koechlin. Protein in clear solutions was also estimated with a Beckman Model DU spectrophotometer by measurements of optical density a t 280 mp. The following extinction coefficients were used for calculation of mass of protein: for crystalline bovine serum albumin, (at 280 mp) = 6.6 grams per 100 sq. cm.; for electrodialyzed amorphous bovine insulin in 0.05 X hydrochloric acid, (at 280 mp) = 10.0 grams per 100 sq. em. (determined by D. F. Kaugh); for human serum albumin, (at 280 mp) = 5.32 grams per 100 sq. cm. PROCEDURE AND RESULTS
Dry crystalline bovine serum albumin was weighed and dissolved in distilled water t o give a concentration of 7.0 mg. per ml. This solution was dialyzed and then diluted to an estimated concentration of 2.5 mg. per ml. (Solution A); measurement of optical density a t 280 mp indicated a concentration of 2.49 mg. of protein per ml. of dialgzed solution. Two-milliliter aliquots were dried and weighed under the following conditions (see Table I): direct drying at atmospheric pressure, followed by cooling in room atmosphere, drying a t atmospheric pressure, followed by cooling in a desiccator, or drying in a vacuum oven and cooling in a desiccator. Tim-milliliter aliquots were also precipitated with trichloroacetic acid a t room temperature, and the residues dried a t atmospheric pressure and cooled in a desiccator before weighing (Table I, column 4). To demonstrate the contribution of the absolute error of the balance to the variation in weights, a second solution of bovine albumin containing approximately 12.5 mg. per ml. was dialyzed (Solution B); spectrophotometric estimation indicated the content of protein to be 11.96 mg. per ml. of dialyzed solution. Two-milliliter samples were dried a t atmospheric pressure and cooled by exposure to room atmosphere (Table I, Solution B, column 5). To demonstrate the effect of the presence of sodium chloride upon dry weights of proteins after trichloroacetic acid precipitation, a third solution of bovine albumin containing 2.5 mg. per ml. was dialyzed (Solution C); spectrophotometric estimation indicated a protein content of 2.58 mg. per ml. of dialyzed solution. A number of 2.0-ml. aliquots were simply dried (Table I, Solution C, column 6), dried after precipitation with trichloroacetic acid (column 7), and dried after precipitation with trichloroacetic acid in the presence of 2.5 mg. of sodium chloride per 2.0-ml. sample (column 8). All weights in Table I are minimum weights, attained in 48 hours in columns 1, 2, and 3 ; in 72 hours in column 4; in 24 hours in column 5 (Solution B ) ; and a t varying times up to 96 hours in the remaining series. In almost all samples there was an increase in weight after minimum weight had been achieved, usually varying from 0.1 to 0.3 mg. but occasionally amounting to 0.8 mg. Electrodialyzed, dry, amorphous, bovine insulin was weighed and dispersed in distilled water; the final concentration was 2.5 mg. per ml. The protein content of an aliquot of the dry material, dissolved in 0.05 N hydrochloric acid, was also determined by measurement of the optical density a t 280 mp; the concentration determined by weighing was 0.450 mg. per ml., and by optical density was 0.460 mg. per ml. Two-milliliter dialyzed samples suspended in water were dried at atmospheric pressure. Other aliquots of this preparation were preripitated
Table I. 1" 5.1 3.9g 4.9 4.9 5.1 4.9 5.0 5.4 4.9 5.1
Determination of Crystalline Bovine Serum Albumin Solution A, Mg. solution B, M ~ . Solution C, Mg. 30 4.4 4.9 4.9 5.0 4.9 5.1 5.1 5.1 5.0 4.8 4.9 4.5 4.6 5.0 4.9 0.19 0.24 3.8 5.0 2b 4.8 5.0 4.8 5.3 5.1 5.0 4.9 4.6 5.1 5.0
4d 5.2 4.9 4.8 5.0 4.6 4.9 4.6 4.5 4.5 4.4
5b
6b
24.5 24.0 24.2 23.9 24.6 24.2 24.3 24.4 24.6 24.7
;,!
a.J 5.1 5.0 4.9 5,0 4.8 5,3 4.9 5.6
76 6.70 5.0 5.0 5.1 5.3 5,2 5.4 5.6 5.4 6.2
81
5.4 5 7 5,7 5.2 5.7 5 2 5.1 5.0 5.2 5.7
Mean 4 . 9 4.7 24.3 5.1 5,5 5.4 S.D. 0.39 0.26 0.27 0.27 0.57 0.29 C.V.% 8 . 0 5.5 1.1 6.3 10.4 5,4 Meanh 5 . 0 5.4 S.D.h 0 . 1 4 0.38 C.V.%h 2 . 7 7.0 Dried a t atmospheric pressure, cooled in desiccator. b Dried a t atmospheric pressure, cooled by exposure t o room temperature. C Dried in vacuo, cooled in desiccator. d Precipitated with 2.0 ml. of 20% trichloroacetic acid a t 23O, dried a t atmospheric pressure, cooled in desiccator. Precipitated with 2.0 ml. of 20% trichloroacetic acid a t 23O, dried a t atmospheric pressure cooled by exposure t o room temperature. f Solution C with 6dded NaCJ, 2.5 mg./2 ml., precipitated with 2.0 ml. of 20% trichloroacetic acid a t 23 washed with 2.0 ml. 10% trichloroacetic acid, dried a t atmospheric p r e s h r e , cooled by exposure t o room temprrature. 0 If these values are omitted, statistics marked h result.
with trichloroacetic acid either a t room temperature or a t 90" C. and the residues were similarly dried. Results are presented in Table 11. The same procedures were followed with a solution of human serum albumin (Cutter Laboratories) diluted from the stated concentration of 25.0 mg. per ml. to a concentration of 2.5 mg. per ml. The concentration of protein was also determined by measurement of absorption of ultraviolet light; a sample volumetrically measured to contain 0.500 mg. of protein per ml. was found to contain 0.493 mg. per ml. on the basis of absorption spectrophotometry. The nitrogen content of the concentrated solution of albumin was 16.8 grams per 100 grams of protein. Dry weights Fere measured after procedures similar to those performed on insulin: results are shown in Table 11.
Table 11. Determination of Electrodialyzed Amorphous Bovine Insulin, Human Serum Albumin, and Pituitary Extract *
Treatment 1.
Direct drying
2.
Precipitation with 2.0 ml. of 20% trichloroacetic acid a t 900 c. Precipitation with 2.0 ml. of 20% trichloroacetic acid a t 230 c.
3.
Insulin 3.4 4.1 3.4 3.2 3.5 3.3
Human Serum Albumin 5.2 4.9 4.7 4.9 4.7 5.2 5.4 5.3
Pituitary Extract 6.5 7.3 6.9 6.0 6.7 6.5
Crude dry pituitary extract was weighed and dispersed in water; the final concentration was 5.0 mg. per ml. The suspension was dialyzed, and 2-ml. aliquots were dried or precipitated with trichloroacetic acid and the residues were dried. The dried precipitates obtained by treatment with trichloroacetic acid a t 90" C. were extracted with 10 ml. of ethyl ether after 72 hours of drying and weighed again after 3 hours of drying; no change in weight was noted. Results are shown in Table IT: Here minimum weights are presented; they were attained in 48 hours for most of the insulin samples, in 24 hours for the albumin, and in 48 hours for the pituitary extract samples. These samples alsoshowed increase of weight up to 0.8 mg. after minimumweight had been achieved. Sodium chloride was added to solutions of human serum albumin in varying proportions, and precipitation with trichloroacetic acid was carried out a t 90" C. The precipitates were
319
V O L U M E 2 5 , NO. 2, F E B R U A R Y 1 9 5 3 Table 111. Determination of Human Serum Albumin Albumin Mg. M1. 2.5 1.0
10.0
2.0
Mg.
Ml.
Dry Weight, 48 Hours, Mg.
4.0
1.0
2.2
10.0
2.5
2.3
9.6 8 .7 9.2 9.5 9.6 9.0
NaCl
4.0
1.0
10.0
2.5
20.0
5.0
1.8
washed twice with 2 ml. of 10% trichloroacetic acid. are shown in Table 111.
Results
DISCUSSION
The time in which minimum weight of dried proteins was achieved could not be predicted, and had to be determined for each probin and for each procedure. Dried human serum albumin reached minimum weight at 24 hours; dried bovine serum albumin precipitated with trichloroacetic acid reached minimum weight only a t 72 hours. The dry weights of the aliquots of proteins treated by procedures other than these two usually reached a minimum value at 48 hours; insulin and bovine berum albumin showed some variation. A series of bovine serum albumin samples containing 25 mg. were precipitated with trichloroacetic acid and dried. The results are not reported in detail here, but the precipitates in the conical centrifuge tubes formed hard discrete pellets which decreased continuously in weight up to 72 hours. Crushing of these pellets a t that time was followed by further decrease in weight up to 120 hours, when weighings were discontinued. Proteins precipitated and dried while spread over large surfaces reach minimum weight more rapidly (4, 5, 7 ) . Almost all samples of dried proteins showed a definite increase in weight when drying was continued after the minimum had been attained; the cause of this increment in weight has not been demonstrated, but it may be due to the formation of carbonates or oxides. The dry weights observed after precipitation with trichloroacetic acid of bovine serum albumin, of bovine insulin, of crude pituitary extract, and of human serum albumin with or without added sodium chloride all agree well with the weights obtained after direct drying of the salbfree proteins. They also agree with the weights of proteins determined by direct gravimetric and volumetric measurements, by determinations of content of nitrogen, and by determinations based on optical densities of clear solutions. The data on crystalline bovine serum albumin in Table I show excellent recovery of protein in the various procedures used as compared with spectrophotometric determinations, and the mean weights of protein for Solutions A and C are not significantly different statistically. The error inherent in the balance %-as 1 0 . 2 mg., corresponding to 4% for 5-mg. samples, and to 0.8% for 25-mg. samples. The coefficient of variation for the series of 5-mg. samples directly dried are 8.0% for column 1 (2.7% if one conspicuously low sample is omitted), 3.8% for column 2, 5.0% for column 3, and 5.3% for column 6 (Solution C); for a series of 25-mg. samples (column 5, Solution B) it is 1.1%. These values are very close to the stated error of the balance, and differences are probably due to variations in the volumetric delivery of aliquots. The highest precision was attained by drying samples at atmospheric pressure a t 110' C. with subsequent cooling by exposure to room atmosphere without the use of a desiccator. Gravimetric estimation of proteins after trichloroacetic acid precipitation and drying of 5-mg. samples showed coefficients of variation of 5.5% (column 4), 10.4% (column 7), and 5.4%
(column 8). These errors include the error of the balance, volumetric errors, and possibly variations in quantitative precipitation of proteins and retention of trichlaroacetic acid. Precipitation of proteins with trichloroacetic acid and subsequent drying of precipitates a t 110" C. for 24 to 72 hours thus allow gravimetric determination of proteins, with accuracies and recoveries as described above. The mechanism of precipitation of proteins by trichloroacetic acid has not been demonstrated conclusively, and it has been claimed that some proteins are not precipitated by this agent. The formation of an insoluble salt, with the protein as cation, has been hypothesized on the basis of data showing precipitation in trichloroacetic acid solutions only a t p H values lower than the isoelectric point of the protein (1). Quantitative precipitation of the proteins of serum by effective concentrations of trichloroacetic acid between 2 and 22% has been reported (10, 11, 23); the effective concentration of trichloroacetic acid used in the piocedures here described varied between 10 and 13%. Peptides are not precipitated by concentrations of trichloroacetic acid up to 10% (13); however, it has been suggested that 16% trichloroacetic acid may precipitate peptides obtained from hemoglobin after digestion with cathepsin (2). It has been claimed that hydrolysis of proteins is produced by concentrations of trichloroacetic acid up to 22% (16), but this has been disputed (10). I n the experiments here reported, there is no indication of significant hydrolysis of proteins or of precipitation of degradation products of proteins. The dependence of the molecular weight of insulin upon pH, concentration, and temperature has been noted (12, 26), and data have been presented to show that the molecular weight reached a minimum value of approximately 12,000 a t pH 2.0 to 3.0, a t increasing dilutions of insulin, or as temperature was increased. Under the conditions of the present experiments, the p H of the trichloroacetic acid solution was calculated to be 1.2 or slightly higher, and the concentration of insulin was approximately 0.1 gram per 100 ml. Based on the data of Gutfreund (12), the molecular weight of insulin should then have been approximately 12,000. This fact is of interest in assessing the influence of molecular weight on the precipitability of proteins by trichloroaretic acid. Cytochrome e, with a molecular weight of approximately 13,000, is soluble in 1.25% trichloroacetic acid ( 1 7 ) . The precipitability of these two proteins by trichloroacetic acid ~vouldtherefore appear to depend upon factors other than molecular weight. Sodium chloride when present in amounts one half the weight of protein does not affect the estimation of bovine serum albumin after precipitation with trichloroacetic acid (Table I, column 8). Increase of the relative amount of sodium chloride to eight times the weight of human serum albumin, as shown in Table 111, may produce an average deficit in weights of protein after trichloroacetic acid precipitation of 13.7% for 2.5-mg. samples, and of 7% for 10-mg. samples. Lipides are best extracted by procedures on moist, fresh precipitates as described by Schneider (22) and applied by others (65). Trichloroacetic acid decomposes a t 110" C. to chloroform and carbon dioxide (10, 13, 21). I t has been reported (8, 9) that precipitated serum proteins in the moist state retain 0.550 mg. of trichloroacetic acid per gram of protein; a great part of this retained trichloroacetic acid 'CI as eliminated by drying of the precipitates a t 105' C. for 24 hours, but the weights of these precipitates were found to represent 108% of the weights of serum proteins precipitated by acetone or alcohol and similarly dried. These data were interpreted to indicate retention of trichloroacetic acid by dried precipitates of protein. However, quantitative precipitation of proteins by acetone or alcohol was not proved, and it is possible that drying of proteins precipitated with trichloroacetic acid was not continued for a sufficiently long time or over an adequate surface to bring about complete elimination of trichloroacetic acid. The studies here reported do not indicate
ANALYTICAL CHEMISTRY
320 consistent retention of trichloroacetic acid by dried precipitates of proteins, although some of the series (Table I, columns '7 and 8; Table 11, No. 3) show mean minimum weights approximately 4 to 8% in excess of the amounts of protein determinrd hy direct drying or by spectrophotometric methods. Gravimetric determination of proteins dried without other treatment a t temperatures between 80' and 110" C. has been reported (3, 6-7, 18-20, 2 4 ) ; such weights have been stated to represent anhydrous weights of proteins (3, 6, 6). I t is possible that residual water may ]cad to errors in the estimation of drv weights of proteins The weights obtained in these studies after precipitation of proteins with trichloroacetic acid are consistent and reproducible and may represent anhydrous weights; however, no attempt has been made t o investigate the dried residues for content of water.
Daranyi, J. v., and Gozsy, B. v., Biochem. Z., 239, 110 (1931). Duliere, W,L., Bixhem. J . , 30, 770 (1936). Duliere, W ,L., and Minne, R., Compt. rend. soc. b i d , 125, 1040 (1937).
Greenwald, I., J . Bid. Chem., 21, 61 (1915). Ibid., 3 4 , 9 7 (1918). Gutfreund, H., Biochem. J . , 4 2 , 544 (1948). Hiller, 4.,and van Slyke, D. D., J . Biol. Chem., 53, 253 (1922).
Hoch, F. L., and Tallee, B. L., J . Bid. Chem., 195, 531 (1952). Hoch, F. L., and Vrtllee, B. L., J . Clin. Invest. (Proc.), 3 0 , 650 (1951).
Hofman, A I . , and Richter, .1.F., &usripis deskosbv. Ldkdmictua, 1 9 , 5 1 (1939).
Keilin, D., and Hartree. E. F. Pior. Roy. SOC.London, B122, 298 (1937).
Kirk, P., Aduunces in Protritz C'henl., 3 , 139 (1947). Knipping, H. W., and KoTits, N. L.. 2. physiol. C'kem., 135, 84 (1924).
BlcMeekin, T. L., and Warner, R. C., J . .4m. C'hem. Soc., 64, ACKNOWLEDGMENT
I t is a pleasure to acknowledge the advice and aid rendered I JDavid ~ F. Waugh of the Department of Biology, Massachusetts
Institute of Technology.
2393 (1942).
Neuberg, C., and Strauss, E., J . Ezptl. M r d . S u r g . , 3, 39 (1945). Schneider, W. C., J . Bid. Chem., 1 6 1 , 2 9 3 (1945). Schofield, R. K., and Samuel, L. W., Suture, 134, 665 (1934). Starlinger, W., and Hartl, K., Biochem. Z.,160, 113 (1925). Strittmatter, C. F., and Ball, E. G., Proc. Nutl. Acud. Sci., 3 8 , 1 9 (1952).
LITERATURE CITED
.ibraham, L., Bull. soc. chim. b i d , 2 , 7 5 0 (1938). (2) Anson, M. L., J . Gen. Phusiol., 2 3 , 695 (1940). (3) Barnett, C. W., .Jones, R. B., and Cohn, R. B., J . E x p t l . hled., 5 5 , 6 8 3 (1932). ( 4 ) Barrett, E.,A m . J . Clin. Path., Tech. Suppl., 9 ( I I ) , 3 (1939). (5) Brand, E., and Kassell, B., J . B i d . Chem., 145, 365 (1942). (1)
(15) Cohen,
E. J., and Edsall, J. T., "Proteins, -4mino Acids and Peptides as Ions and Dipolar Ions," p. 376, S e w York, Reinhold Publishing Corp., 1943.
Tietze, F., and Neurath, H., J . Bid. Chem., 194, 1 (1952). Vallee. B. L.. and Hoch. F. L.. J Clin. Inliest. ( P r o c . ) , 2 9 , 850 '(1950). RECEIVEDfor review February 20, 1952. .4ccepted October 15, 1952. Work supported by a grant-in-aid from the Charles R. Blakely Fund for Research on Lymphatic Leukemia of the Division of Medical Sciences, National R~searchCouncil, and performed when F. L. Hoch waa a postdoctoral research fellow of the United States Public Health Service, and B. L. 1-allee was a senior research fellow of the National Research Council Committee on growth, supported by the -4merican Cancer Society.
Flame Photometric Determination of Calcium, Strontium, and Barium in a Mixture ORVILLE N. HINSVARK, SYLVAN H. WITTWER, AND HAROLD M. SELL Departments of Horticulture and Agricultitral Chemistry, Michigan State College, East Lansing, Mich. A flame photometric procedure is presented for the determination of the alkali earth carbonates in a mixture. When the carbonates are dissolved in a perchloric acid solution, strontium interferes with calcium and calcium interferes with barium at the wave lengths employed. By subtracting a linear correction from the emission intensity readings, it is possible to obtain accurate results, rapidly and simply, without separation.
T
HE use of flame photomrtric procedures has received wide attention in recent years for the determination of sodium, potassium, magnesium, and calcium in plant tissues (2, 3 ) . Studies in this laboratory concerned with the absorption and utilization of the alkaline earths by plants has necessitated a rapid and accurate estimation of small quantities of calcium, strontium, and Ilarium individually and in mixtures. By using a flame photometer, a method has been found foi the determination of each in a mixture, whereby a correction for the interferences due to thr other8 is applied. EXPERIMENTAL
The flame intensity data were obtained by employing a Beckman DU spectrophotometer with acetylene flame attachment (No. 9200). The carbonates of the metals were used, because they could be separated with relative ease; and by dissolution in perchloric acid, the anion effects ( 1 ) could be minimized. Baker's analyzed reagent grade calcium carbonate, barium carbonate, and perchloric acid (70%), and Mallinckrodt's analytical reagent grade strontium rarbonnte were used.
Stock solutions of the individual metals were obtained by dlssolving a weighed quantity of the dried carbonate ( 1 gram per 100 ml.) in the required volume of 10% (by volume) perchloric acid. The reference curve for each metal was obtained by making flame intensity measurements on solutions of known concentration, prepared by taking varying quantities of the stock solution and diluting to 25 ml. To ascertain the interference of one alkaline earth on another, a constant amount of one stock solution was introduced into a number of 25-ml. flasks. Varying volumes of another stock solution were added to these flasks and the solutions were made to 25 ml. Flame intensity measurements were made on these solutions and the change in emission intensity, due t o the interference, mas measured. This was done for each alkaline earth in combination with the other two; and if one ion interfered m-ith the other, the flame intensity was found to increase in direct proportion to the concentration of the interfering ion. The intensity of the observed line was read on the transmittancy scale using the red-sensitive phototube with selector switch set at 0.1. The wave lengths chosen were, as read off the instrument dial: (1) calcium = 623 mp: (2) strontium = 681 mp; and ( 3 ) barium = 871 mp. These lines were chown t)rc:Luv the intensities were comparable to those more generally
