ANALYTICAL CHEMISTRY
160 and Seibert, C. B., U. S. Bur. Mines, Reptr. Invest. 4782 (1951). (96) Stratton, R. C., Ficklen, J. B., and Krans, E. IT., IND.ENG. CHEM., ANAL.ED.,4,334 (1932). (97) Sweeney, 0. R., Outcault, H. E., and Withrow, J. R., J . Znd. Eng. Chem., 9,949 (1917). (98) Taylor, E., and Johnstone, H. F., IND. ENG.CHEM., ANAL.ED., 1,197 (1929). (99) Thomas, M. D.,Zbid., 4,253 (1932). (100) Thomas, M. D., private communication. (101) Thomas, M. D., and Abersold, J. N., IND. ESG. CHEX, .ANAL. ED.,1, 14 (1929). (102) Thomas, M. D . , and Cross, R. J.,Ind. Eng. Chem., 20, 645 (1928).
(103) Thomas, B.I. D., Ivie, J. O., and Fitt, T. C., IND. ENG.CHEM., ANAL.ED., 18, 383 (1946). (104) Tolman, R. C., Reyerson, L. H., Brooks, A. P., and Smyth, H. D., J. Am. Chem. Soc., 41, 587 (1919). (105) Tolman, R. C., Reyerson, L. H., Vliet, E. B., Gerke, R. H., and Brooks, A. P., Ibid., 41, 300 (1919). (106) Tolman, R. C., and W e t , E. B., Zbid., 41, 297 (1919). (107) United States Technical Conference on Air Pollution, “Air Pollution,” New York, McGraw-Hill Book Co., 1952. (108) Crone, P. F., and Boggs, W. E., ANAL.CHEM.,23, 1517 (1951). (109) Velten, H. J.,Znd. Eng. Chem., 29, 1214 (1937). (110) Weber, H. C., Ibid., 16, 1239 (1924).
RECEIVED for review June 27. 1951.
Accepted October 21, 1952.
Photonephelometric Microdetermination of Sulfate and Organic Sulfur GERRIT TOENNIES AND BOHDAN BAKAY Institutefor Cancer Research and Lankenau Hospital Research Institute, Philadelphia 11, Pa. Because of the need for a reliable, simple, and precise method for the determination of microgram quantities of sulfur, the nephelometric measurement of sulfate has been re-examined, with the aid of a photoelectric instrument. An analytical method has been developed which is characterized by the use of a glycol-ethyl alcohol-water system as a means of obtaining reproducible and stable suspensions of barium sulfate. The method permits determinations with a precision of i l % on samples containing 3 micrograms of sulfur-i.e., approximately one hundredth of the minimum amount required by current micromethods. Ions like ammonium, sodium, potassium, calcium, ferric, chloride, nitrate,or phosphate cause no practical interference even when present at high concentrations relative to sulfate. The method is adaptable to sulfate concentrations ranging from 0.01 to 100 p.p.m. Its application to the microdetermination of sulfur in organic substances is described.
A
MONG the numerous possibilities which have been con-
sidered for the determination of sulfur, procedures centering around precipitation of barium sulfate continue to command the widest acceptance. A high degree of specificity in acid solutions, a high degree of insolubility, and a favorable gravimetric factor are the main advantages of barium sulfate. I n order to utilize the advantages of barium sulfate in the microgram range of microanalysis, measuring methods other than weighing must be resorted to. So far, titrimetric methods using tetrahydroxyquinone (THQ) or rhodizonates as indicators have proved of greatest practical usefulness for this purpose. Recent publications ( 2 , 17) show that a practical sensitivity of =t3 micrograms of sulfur per determination can be attained in the titration of sulfate with barium chloride when tetrahydroxyquinone serves as indicator and the comparator technique of Ogg, Willits, and Cooper ( 1 4 ) is used for end-point definition. Accordingly, with that technique, a determination precise to ilY0 requires 300 micrograms of sulfur. The method here described increases this level of analytical sensitivity up to 100-fold-i.e., as little as 3 micrograms of sulfur can now be determined with similar precision. The determination of barium sulfate in suspension by measurement of light absorption (turbidimetry) or light scattering (nephe-
lometry) has been studied previously, primarily in connection with clinical or technical measurements. Among the best documented procedures of this type are those of Denis and Reed (‘i‘), Sheen, Kahler, and Ross (15), Medes and Stavers ( f d ) , and Nalefski and Takano (13). The apparent sensitivities of these methods range from 0.002 to 0.4 microgram of sulfur per ml. of suspension. Despite their high sensitivity, none of these methods has gained acceptance among scientific analysts, presumably because in practice the stability and reproducibility of the barium sulfate suspensions used were found to be poor and good measuring instruments were not readily available. The authors became interested in the possibilities of nephelometric sulfur determination because of the need, arising from research on nucleoproteins, for a method which could precisely determinefractionsof 1%of sulfur in IO-mg. samples of substances which contain 4 to 5% of phosphorus. Disappointing results, as regards reproducibility and stability, with precipitates obtained in aqueous sodium chloride hydrochloric acid solution (13, 15) and measured in the Coleman photonephelometer led to an investigation of the analytical possibilities of truly colloidal suspensions of barium sulfate obtained in semiorganic media. I n 1922 Bechhold and Hebler ( 4 ) ,studying the effects of alcohol and glycerol on the colloidal properties of barium sulfate sols, found that a t low sulfate concentrations alcohol promotes precipitate formation while glycerol acts as a colloid stabilizer. The present experimental attack, which was based on those findings, led to the results here described. EXPERIMENTAL
General Technique. 811 measurements were made in lipless Pyrex (Corning Catalog S o . 9820) 19 X 150 mm. test tubes, with the Coleman KO. 7 photonephelometer. The cuvette holder of the instrument was equipped with a sleeve insert made of Plexiglas ( 3 ) and tubes were selected to be held snugly by this sleeve. For further selection the tubes were filled with a barium sulfate suspension prepared in bulk, and only those whose readings fell within a 4~0.5%range and were independent of changes in rotational position were retained. The instrument is operated according to instructions. (which include filling the outer space of the cuvette holder with water). By manipulation of the knobs the sensitivity of the instrument can be varied over a 200-fold range. As shown below, all parts of the sensitivity scale can be utilized for sulfate determinations, but most of the experimental measurements were made a t the standard sensitivity, defined by the Coleman nephelos standards, which differs by a factor of approximately 14 from the highest and the lowest sensitivity.
161
V O L U M E 25, N O . 1, J A N U A R Y 1 9 5 3
even the purest grades of glycerol contain enough sulfate to produce nephelometrically measurable amounts of barium sulfate when added, with alcohol, to clear filtered aqueous barium chloride solution. Thus the alcohol-glycerol barium chloride reagent is "seeded." Even when working with a single glycerol preparation, the colloidal characteristics of this barium sulfate seed cannot be consistently reproduced from one operation to another and its properties change with time. ;1reagent used immediately after its preparation, before thr seeds have had time to form, tends to produce barium sulfate of bluish opalescence, associated with high nephelodensity. Linearity of response under these conditions is poor, as the bluish precipitate changes on standing, and a t the lower concentrations its formation is delayed. After the reagent has stood for several hours it will produce barium sulfate of a grayish opalescence and lower nephelodensity. It is in this condition, which may last from 5 to 30 hours after preparation of the reagent, that linearity of response is a t its best. As the reagent gets older, the seeds apparently aggregate, and the resulting precipitates vary in optical characteristics, so that responses are erratic. At the higher concentrations the beginnings of crystallinity with attendant settling develop sooner than a t low concentrations, so that the pattern of response tends to be convex.
ETHANOL
7 90
I) -r
9 9w
60
q A
30
t 7 GLYCEROL
20
40
60
ORGANIC SOLVENT IN MEDIUM, VOL. %
Figure 1. Effect of Organic C o m p o n e n t s on Nephelodensity of B a r i u m S u l f a t e 2 X 10-5 M K ~ S O P0.02 , M BaCln, 0.1 M "01 90
Exploratory experiments soon showed that in order to obtain identical nephelos readings among several tubes of identical contents prepared individually but in a single operation it is necessary to combine equal volumes of the two component solutions (sulfate and barium ion), which must be identical R-ith regard to solvent composition. The temperature of the component sohtions, within a 10" range, the sequence of addition (barium to sulfate or vice versa), and the speed of addition have little or no effect,however. The contents of the t u b e are mixed by swirling and readings arc taken after 10 to 20 minutes of undisturbed standing. Because colloidal barium sulfate forms a fine adhering film, tubes must be carefully brushed with detergent solution and thoroughly rinsed before re-use. It has also been found necessary, as ft further protection against contamination, to autoclave all tubes and other critical containers in sulfate-free v.-ater (see Reagents) a t 120' C. (18). Effect of Organic Components of Medium on Nephelodensity of Barium Sulfate. Experimentation with aqueous media containing ethyl alcohol and glycerol led to the findings shown in Figure 1. Ethyl alcohol produces densities which are high but a t the same time highly dependent on alcohol concentration and prone to quick settling, while glycerol by itself has little effect. However, the use of a 2 to 1 mixture of the two agents produres colloids which are stable, while their nephelodensity is, over a fair range, independent of the concentration of the mixture. Therefore, this mixture, a t the 407, level, was used for further experimentation. Factors Determining Reproducibility and Linearity. Under the conditions selected barium sulfate precipitates in graded concentrations, up to 8 X 10-5 M , yielded turbidities linearly proportional to quantity in many instances, while in many cases responses were either erratic or curved. The lengthy experimental studies undertaken to overcome these difficulties, and the resulting conclusions, may be summarized as follows: The most important determinant of the nature of the barium sulfate precipitate is the sulfate content of the barium chloride precipitating reagent. In working with solutions of barium chloride in aqueous alcohol-glycerol mixtures, it was found that
2
t
8 80 -1
Y
4 Z
70
I1 I
1
I
0.08
0
I 0.16
I
0.32
BARIUM CHLORIDE MOLAR CONCN.
Figure 2.
Nephelodensity of B a r i u m Sulfate (4 x 10-6 M )
Formed by unacidified and sulfate-free barium chloride reagents of different ages and concentrations. Sulfate solution and reagent* contain a0 vol. % of 9 to 11 ethyl alcohol-di ropylene glycol mixture. Sulfate solution contains 0.15 M "01. b m e s in minutes shown on curves indicate periods elapsed between preparing reagent (by combining filtered aqueous barium chloride solution with organic mixture) and adding it to sulfate solution. Each point was determined in triplicate.
Attempts to make the reagent sulfatefree by filtration or centrifugation failed, but replacement of glycerol by another glycol led to the solution. Available data (6, 9) and further experimentation led to the use of a 9 to 11 mixture of ethyl alcohol and dipropylene glycol, which proved to resemble the ethyl alcohol-glycerol mixture in colloid-protective action while being free of the difficulties caused by sulfate contamination.
ANALYTICAL CHEMISTRY
162 Although dipropylene gl>-col is free of measurable quantities of sulfate, barium sulfate seeds or aggregation embryos (11) still appear t o play an important role as determinants of the colloidal pattern. This is evident from the experiments described in Figure 2. It is obvious that a t the lower barium chloride concentrations the propel ties of the reagent undergo a rapid change during the first few hours. That the rate of this change increases with barium concentration is consistent with the assumption that formation of latent harium sulfate occurs. Since the process seems to be nearly coniplete within 5 minutes when 0.25 31 barium chloride is used (0.125 -11 final concentration), the attempt wc~s made to use this concentration for the precipitation of graded amounts of sulfate. I t was abandoned, however, because, over the range of 2 t o 8 X 10-6 J4 sulfate, the responses, while homogeneous, varied from series to series, concave and linear, as well as conves curves being obtained. \Then, instead, a barium chloride concentration of 0.04 *?I was adopted and the reagent was not used until 2 hours after its preparation, linear responses were obtained consistently. This reagent is good for approximately 40 hours. K h e n it gets older, responses tend to be low a t the upper sulfate levels, resulting in a slightly conye\ response curve or erratic points.
90
*
In t
8z 6 0
on the barium sulfate reading. I n order to extend this observation, identical amounts of ammonium sulfate were dissolved in the usual medium, by itself, and in the presence of 10-fold molar quantities of each of the following salts: ammonium chloride, disodium hydrogen phosphate, potassium chloride, calcium chloride, and ferric chloride. The results are shown in Figure 4. The slightly higher values obtained in the presence of the salts can he attributed largely to sulfate impurities. In further esperiments a 1000-fold quantity 0:‘ ammonium nitrate or a 1000fold quantity of sodium nitrate showed no significant effect. The evidence thus indicates that ammonium, sodium, potassium, calcium, ferric, chloride, nitrate, and phosphate ions have no practical influence. Minimum Sulfate Level. For the standard instrument setting the range of measurement is from approsimately 0.9 to 9 X .M sulfate. Utilization of the maximum sensitivity of the instrument, which is approximately 14 times greater than the standard sensitivitj-. ~ o u l dlower the upper concent.ration limit JI sulfate. Even if the solubility-deto less thaii 0.7 X pressing effect of the organic. components is disregarded, this concentration still is 250 times as high as the solubility of barium sulfate in the presence of 0.02 111 barium ion. However, a t these low concentrations the formation of barium sulfate, which can no longer be distinguished by the eye, is slow and uncertain. I n order to make measurement of the smallest amounts of sulfate practical it is necessary to provide a base or blank level of SUIfate of approsimately 0.9 X 10-5 Jf and to measure the increase in barium sulfate resulting from the unknown. When operating a t maximum sensitivity, the instrument should be so adjusted that a t the blank level of 0.9 X IO+ 91 barium sulfate it reads well below 50. Maximum Sulfate Level. Use of Spectrophotomster. Sdjustment of the photonephelometer to minimum sensitivity permits extension of measurements up to a barium sulfate concentration of approximately 1.25 X M. The same con/
4
Y
SULFATE STANDARD PLUS l o x AS MUCH (&IOF: KC1, CaC12,
8 Z
30 90
* I
0.037
I
0.075
1
0.15
t In
B
8 60
NITRIC ACID MOLAR CONCN.
J
Figure 3. Nephelodensity of Barium Sulfate at Different Nitric Acid Concentrations
B
Y
z
6.4 X 10-5 M in upper curve, 3.2 X 10-5 M i n lower curve. 40 vol. yo of 9 to 11 ethyl alcohol-dipropylene glycol, 0.02 M BaClr
In these experiments and in the final procedure all acid was incorporated into the sulfate solution while the barium sulfate precipitating reagent was left neutral. This was done because, unlike the situation with regard to organic components, no advantage accrues from distributing the acidity equally between sulfate and reagent, and because concentrating it in the sulfate solution lends greater flexibility t o the method when it is applied to acid solutions. Effect of Acidity. Figure 3 shows that the acidity level selected, 0.075 M , is well within a range where small variations are of little consequence. Effect of Other Ions. Early experiments showed that phosphate in 10 times the molar quantity of sulfate has no influence
30
15
30
45
60
- r
13
AMMONIUM SULFATE, MM.
Figure 4. Nephelodensity of Barium Sulfate in Presence of Other Ions 40 vol. 70of 9 to 11 ethyl alcohol-dipropylene glycol, 0.075 M “01. 0.02 W BaClp
V O L U M E 2 5 , N O . 1, J A N U A R Y 1 9 5 3
163 Volatility of Sulfuric Acid. Fifteen-milliliter aliquots of a 9 X 10-5 sulfuric acid solution in sulfate water (see Reagents) were evaporated on the steam bath (a)just to dryness, and ( b ) with heating on the steam bath continued for 3 hours after dryness had been reached. The residues were dissolved in 25 ml. of a mixture containing the proper amounts of the organic components and nitric acid in sulfate-free water (see Reagents). Another aliquot (aa)of the same sulfuric acid solution was brought to the same final concentration without prior evaporation. Solutions (a)and (b) shoir-ed 96.7 and 88.7%, respectively, of the sulfate value of solution (aa).
150
f
5
;100 4
u
B
50
I
-300
I
I 500
I
I
I
700
WAVE LENGTH, M r
Figure 5. Absorption Spectra of Barium Sulfate Suspensions Prepared under Standard Conditions 4 X 10-5 M i n upper, 3 X 10-5 M in lower curve Coleman .Model 11 spectrophotometer
centration range can be measured by light absorption in the Coleman spectrophotometer. Figure 5 shows absorption spectra of barium sulfate suspensions prepared under the experimental conditions. Calibration curves obtained with two different instruments (Coleman spectrophotometer, Models 11 and 14) a t a wavelength setting of 350 mp--i.e., in the range of maximum absorption-produced linear turbidity responses corresponding to optical densities of 0.0415 (Model 11)and 0.0555 (Model 14)per 31 barium sulfate. -4ccording to the data of Figure 5, 1X use of the range of minimum absorption, a t 700 mp, may permit extension of measurements up to 4 X 10-3 J E barium sulfate. Interfering Combustion Products. The data shomx in Figure 6 are representative of many observations made in the determination of sulfate in combustion products of organic material. Obviously, if two solutions are analyzed which, as far as the analytical method is conrerned, differ only in their sulfate contentfor instance, a sulfate standard and a solution of unknown sulfur content-plotting of the results obtained with different aliquots should result in curves which, like the upper two of Figure 6, converge a t the zero level-Le., the over-all blank value. This, together with linearit). of the responses, is an important criterion in judging the validity of any set of determinations obtained by the present method. Failure to meet this criterion is evidence of contamination in one of the solutions. Experimental evidence, illustrated by Figure 6, indicates that the Parr bomb oxidation product of the organic material used (nucleoprotein) contains undefined factors which give rise to anomalous sulfate values. It became further apparent, and is shown by examples in Figure 6, that these undefined factors tend to disappear on standing and can be eliminated by boiling. The real nature of this phenomenon must remain unexplained a t present. A simple and reliable means of overcoming the difficulties arising from it is to evaporate all combustion products, such as Parr bomb washings or Carius tube washings, to dryness. The necessity of adding a nonvolatile base in this step, whenever free sulfuric acid may arise, is demonstrated in the next section
ALIQUOTS, ML.
Figure 6.
Presence of Interfering Factors in Parr Bomb Combustion Products
300-mg. samples of thymus nucleoprotein preparations oxidized according t o Callan and Toennies (5). Bomb washings diluted t o 100 m l . 1. Sulfate standard 5. Aliquot of combustion product after 18-hour standing 3. Same, after 72-hour standing 4. Same, after 18-hour standing and 1-minute boiling 2. Same, after 25-minute boiling
A similar experiment, in which the solution contained, in addition, 9 X 10-4 M phosphoric acid and 0.01 M sodium nitrate showed no measurable loss of sulfate during 5 hours of drying on the steam bath. The evidence thus indicates that losses arising from the vapor pressure of sulfuric acid a t 100" C. may be significant a t these low quantities even during short heating periods; all evaporations involving free sulfuric acid should, therefore, be conducted in the presence of excess sodium nitrate. 4N.4LYTICA L PROCEDURES
Reagents. SULFATE-FREE \TATER. Distilled water freed of measurable quantities of sulfate by double distillation or by passage through ion exchange resins. The authors allow distilled water to flow by gravity through a column 5 cm. thick and 25 cm. long, of Monobed resin (LaMotte). Depletion of the column is conveniently revealed by a change in the color of the resin, from dark to light, near the entrance of the water. At the slightest color change the resin should be renewed. SULFATE WATER. iimmoniuin sulfate (purest), 3 X 10-6 M, in sulfate-free water (see above). This is not needed if the aqueous solutions to be analyzed contain more than 1 X sulfate.
ANALYTICAL CHEMISTRY
164
NITRATEWATER. Sodium nitrate (purest), 3 X 10-8 M , in aulfate-free water. This is not needed unless solutions are to be analyzed which require evaporation (see Interfering Combustion Products) and which on evaporation would leave free sulfuric acid behind. ETHYL ALCOHOL-DIPROPYLENE GLYCOL MIXTURE(ED MIXTURE). Mix 450 ml. of absolute ethyl alcohol and 550 ml. of dipro ylene glycol (Carbide and Carbon Chemicals Co.). 4 0 4 ED. Mix 400 ml. of E D mixture with 500 ml. of sulfate water, add 150 millimoles of nitric acid, and, after cooling, dilute to 1000 ml. with sulfate water. I n the analysis of solutions M sulfate, sulfate water need containing more than 1 X not be used. Make up all standard solutions in 40% SULFATE STANDARDS. ED. Dissolving of 52.9 mg. of ammonium sulfate (purest) in 250 ml. of 40% E D and dilution of a 10-ml. aliquot to 100 ml. with 40% E D yield the 1.6 X M standard which is used a t the normal sensitivity of the instrument. BARIUMCHLORIDEREAGENT.Prepare 100 ml. of 1.34 J4 barium chloride in ordinary distilled water; after 3 hours (or longer) of standing, filter the solution through a fine (F) sinteredglass filter. This solution may be used for a long time. Whenever reagekt is needed, dilute 6 ml. of the 1.34 M solution in a cylinder t o 120 ml. with sulfate-free water, and add 80 ml. of E D mixture, After 2 to 3 hours of standing (aging) the reagent is ready and may be used for the next 40 hours. Sulfate Precipitation and Measurement. Instrumentation and glassware have been discussed above. For a single analysis, fill 10 calibrated tubes as follows:
40% E D ml Standard'iniO'% ED, ml. Unknown in 40% ED, ml.
1 4 1
2 3 2
3 2 3
4
1 4
Tube No. 5 6 7 0 4 3 5
1
2
9 1
10
2 3
4
5
8
0
Depending on available material and desired precision this series may be simplified-e.g., by omitting tubes 5 and 10, or tubes 2, 4, 7, and 9. To each tube, now containing 5 ml., add 5 ml. of barium chloride reagent in as short a time as possible, using a uniform technique (do not blow, but do not wait for drainage). Mix the contents by sn-irling and measure the nephelodensity of each tube after 15 minutes' standing. It is convenient to plot the readings on precision graph paper. When the points produce straight lines which meet a t the same blank level for both solutions (cf. Figure 6, curves 1 and 2) the determination should be correct. The principle of calculation of results is simply: Sulfur concentration of unknown = sulfur concentration of standard X net nephelodensity of unknown/net nephelodensity of standard. Obviously the result may be calculated from the readings directly by arithmetical procedure, without the use of graph paper. Oxidation of Organic Material. Adaptations of three methods were studied: oxidation in the standard Parr oxygen bomb ( 5 ) , oxidation in the Parr oxygen micro bomb (1, 1 6 ) , and oxidation by the Carius micro method (8). The latter was found to be the most satisfactory method for the present purposes. Complete reagent blanks were run in the case of the two micromethods.
atmospheres. The proper amount of electric current to obtain ignition without burning the platinum spiral was introduced by means of a rheostat (Variac). The correct rheostat setting was determined by prior experimentation. Because of the large heat capacity of the body of the bomb, it gets barely warm to the touch, and immersion in water, as with the macrobomb, is not necessary. After 1 hour the pressure was carefully released, the bomb was opened, 15 ml. of sulfate-free water were added, and the bomb was closed and thoroughly shaken to wash all parts. After reopening 15 ml. were removed and evaporated to dryness on the steam bath. Dissolving the residue in 12.5, 20, or 25 ml. of 40% E D yielded the solution ready for analysis. This novel procedure, which eliminates complicated rinsing operations, gave good results with amino acids but failed with nucleoproteins, apparently because of incomplete combustion, as revealed by the presence of a water-soluble film of oily appearance in the crucible. As the same substances burn well in the macrobomb, the relatively higher heat capacity of the microbomb which results in a shorter and/or lower temperature peak may be responsible. MICRO-CARIES.An electrically heated micro-Carius oven (A. H. Thomas Co., Catalog No. 5713-F) and 13 X 250 mm. Pyrex combustion tubes (A. H. Thomas Co., Catalog No. 5714-A) were used. Five- to 10-mg. samples were weighed out in Pyrex tubes of 5-mm. inside diameter and 20-mm. length, open on both sides. In order to remove traces of sulfate from the glass surface (8), weighing tubes and combustion tubes were treated for 2 to 3 hours with hot fuming nitric acid and autoclaved for 15 minutes with sulfate-free water. The charged weighing tube was placed in the bottom of the combustion tube, 0.40 ml. of fuming nitric acid and 2 drops of concentrated hydrochloric acid were added, and the tube was carefully sealed. Addition of hydrochloric acid (8)was found necessary to obtain complete combustion of nucleoprotein, Tubes were heated for 12 hours a t 250". In order to prevent accidents and losses of combustion products, the cold tube? were immersed in dry ice (IO)to freeze the contents, before opening. After addition of 3 ml. of nitrate water the contents were transferred to a beaker, three additional 3-ml. portions of nitrate water being used for washing. After evaporation on the steam bath and heating until nitric acid odor had disappeared, the residue was dissolved for analysis in 12.5, 20, or 25 ml. of 40Q/, ED. ANALYTICAL RESULTS
Precision. In all determinations solutions of pure ammonium sulfate served as primary standards. Precision was evaluated by comparing graded concentrations of ammonium sulfate a t different sensitivity levels of the photonephelometer and in the range of the spectrophotometer. The respective ranges and conditions are discussed above (see Minimum Sulfate Level and Maximum Sulfate Level). Within each sensitivity range the highest sulfate level was used as the reference standard for a series of lower sulfate levels. These experiments yielded the data recorded in Table I. Table I1 lists in detail the results obtained in the application of Samples of 300 mg. of nucleoprotein S ~ A N D APARR R D BOMB. the method to the analysis of organic materials. The data in of approximRtely 0.3% sulfur were oxidized ( 5 ) . The n.ashings columns 13 and 6 (the latter comparable to the fourth column were made to 100 ml., 10-ml. aliquots were placed in a 25-ml. volumetric flask and boiled gently for 20 to 30 minutes, and after of Table I) give further information on analytical precision and cooling 10 ml. of ED, 3.75 meq. of nitric acid, and sulfate-free show that under practical conditions the average precision is water to volume were added. paRR M ~ -4 ~ ~ 14 mm, ~ inside ~ somewhat~ lower than ~ that obtained ~ in the~direct determination , of pure sulfate (Table I). diameter and 8 mm. high, was used as sample holder. Because the original fiber washer in the gas inlet tube of the bomb seemed to give rise to contamination, it should be replaced by a polyethylene washer. The two filament holders were connected by a Table I. Precision of Sulfate Determinations at Different Levels, spiral of 32-gage platinum wire. A 2-inch cotton Obtained with Ammonium Sulfate thread was hung from the platinum spiral into AV. Sulfate Concentration the sample. The sample (10 to 50 mg.) was mixed Before addition of BRCIZ' with 2.5 mg. of ammonium nitrate by means of a Instrument Final, .%I .M y S/ml. Standard piece of platinum wire which was left in the cruciPhotonephelometer, a t highble. One or 2 drops of Decalin or decane were 6-120 X 10-7 0.02-0.40 12.5 3-60 X 10-7 est sensitivity added to wet the sample. Two milliliters of photonephelometer, at stand1.2-20 X 10-6 0.40-6.0 3~0.5 0.05 -11ammonium hydroxide (in sulfate-free water) ard sensitivity 0.6-10 X 10-5 2-25 X 10-4 6.0-80.0 10.5 were placed in the bottom of the bomb. The Spectrophotometer,35Omp 1-12.5 X 10-4 bomb was closed and charged wit,h oxygen to 45
V O L U M E 25, NO. 1, J A N U A R Y 1 9 5 3
165
Table 11. Results of Photonephelometric Sulfur Determinations on Organic Materials Group of Detps. (1) A
Conditions of .4nalysis Amount Combustion weighed, Substance analyzed method mg. (2) (3) (4) Kucleoprotein I1 Standard Parr 3 1 7 . 8
H
1
Nucleoprotein I I b
J
Nucleoprotein 111
&
hlicro-Cariusd
a
I\.licro-Cariuse hficro-Carius
Weighed a s solution. With reagents as u n a e r S r o u p E. Heated 10 hours a t 220 ,
E
...
2.70 2.47
34.30 40.23
3.25 12.31
0.00025 0 . 0 0 0 2 5 0 00024 0.0304 ... 20.5
...
21.3
99
30.50
4.86
25.64
41.14 30.80 32.26 33.70 28.52 30.70 30.50 30.90
1.70 1.70 1.70 1.70 1.70 1.70 1.70 1.70
39.44 29.10 30.56 31.00 26.82 29.00 28 80 29.20
0 . 120Q
*
...
1.220
Methionine (with Deo- Micro-Parr alin) Reagent blank (“03, Micro-Carius HC1) Leucine Micro-Carius Methionine i12.1 Micro-Carius mg. leupineb Methionine 10.4 hlicro-Cariub mg. leucine Nucleoprotein I Micro-Carius
...
2.057 811 1,540 All 1.613 All 1.685 All 1.426 All 1.535 1.525 All All 1.545 Heated 12 hours a t 270’. Heated 12 hours a t 250’.
D
12.9 s n 10 o 10.1 9.8 10.7 11. 9 11 9
All
Accuracy. For an evaluation of the over-all accuracy attainable by combining oxidation of organic material with photonephelometric determination of sulfate, the amino acid methionine was chosen because it is known to offer difficulties in the complete recovery of its sulfur in many oxidative procedures ( 5 ) . The results of the work with methionine are included in Table 11. The column headings and other details pertaining to Table I1 are based on the procedures described under Sulfate Precipitation and Measurement, and Oxidation of Organic Material. The determinations of Groups C and D (see column 1) show a sulfur recovery of 95 to 97yo when approximately 200 micrograms of methionine were oxidized in the Parr microbomb in the presence of 40 mg. of Decalin. Under these conditions the reagent blank accounts for 6 to 7% of the total sulfur. According to Groups E and F a sample of obleucine contains 0.03% of sulfur. I n Group G the sulfur content of methionine in samples of 60 and 120 micrograms was determined in the presence of a 100- to 200-fold excess of leucine. Recoveries of 95 and 997, of the theory were obtained after subtraction of the over-all blank values (reagents plus leucine). The observed deficits of 1 to 570 in methionine sulfur may be due to volatilization of sulfuric acid since these determinations were made prior t o the experiments reported under ’i‘olatility of Sulfuric Acid. I n Groups H, I, and J are listed the results of replicate determinations on three different nucleoprotein preparations. I n Group H the time and temperature of the micro-Carius oxidation were varied, and because concordant results were obtained, an intermediate level of 12 hours’ oxidation a t 250’ was adopted for further work on nucleoprotein preparations (Groups I and J). Products requiring 16 hours at 290’ have since been encountered. Difficulties due to ( a ) formation of a microcapillary in the seal as a result of gas pressure and ( b ) dilution of the oxidizing mixture by condensation are avoided by sealing after cooling the oxidation tube with a calcium chloride tube attached. Hex-ever, protein substances have been encountered which require even more intense treatment-e.g., 3 drops of hydrochloric acid, and 16 hours’ heating a t 270”. Since the determinations on nucleoproteins I1 and I11 listed under Groups A and B represent aliquots of single oxidations and no blank values were considered, the final values are not directly comparable with the results of the complete duplicates listed under I and J. Limits of Sensitivity. hnalysis of the data of Table I1 indicates that in practical applications the limit of sensitivity lies in the
1 8
...
All
Micro-Parr
...
rs:...,.
All Ail
Decalin blank
Mean Value. &% (13)
...
10.7 0. 060a
C
1/10
% of theory (12)
ti)
15/17 15/17 15/17 15/17 All
Standard Parr 3 1 3 . 8
F
(7, 90.43 86.40 87.78 90.30 82.00 80.15 83.23 2.128 2.500 37.00 42.70 1.64 1.74 1.74 4.95 17.69
AV. Deviation, 70 of
70of substance Av., 7% (10) (11) , . 0.284 0.272 0.276 0.284 0 279 0.261 0.255 0,265 0.260 0.0075 0,0073 0.0074 20.4 200..090 0 2 3 ...
32.0 38.6 0. 191nn 0.2185 700
Nucleoprotein 111
G
y/ml.
(6) 3.617 3.456 3.511 3.612 3.280 3.206 3.329 0.086 0.100 1.850 2.135 0,0822 0,0871 0.0871 0.198 0.7075
B
E
Aliquot analyzed (5) 1/10
Sulfur Found Total Net in In4070 In Blank in ED, aliquot, aliquot. aliquot,
...
1,70 5.38
...
... ..
0.306 0.323 0.306 0.317 0.274 0.271 0.242 0.245
1.5
... 95 97
...
1
1 3
95
2
0.313
...
2.2
0.273
...
0.6
0.243
...
0.6
range of ztO.03 to 0.05 microgram of sulfur (Groups C and E), thus permitting a potential accuracy of =kl% with sulfate samples corresponding to 3 micrograms of sulfur. I n operations involving individual oxidations of organic micro samples and blank subtractions, the practical sensitivity limits increase to 3 ~ 0 . 2to 0.7 microgram of sulfur (Groups D, G, H, I , and J). Causes of Difficulties. If erratic results are encountered, it has in the authors’ experience been possible to trace them to one of three causes. contaminated glassware (discussed under General Technique); the presence of too much sulfate in the sulfate-free water (see Reagents); and deterioration of the barium chloride reagent (discussed under Factors Determining Reproducibility and Linearity). In case of doubt a fresh barium rhloride rragent should be made ( s e ~Reagents). LITERATURE CITED
Agazai, E. J., Parks, T. D., and Brooks, F. R., ANAL.CHEM.,23, 1011 (1951).
Alfcjno, ‘J. F.; Ibid., 20, 85 (1948). Bakay, B , and Toennies, G., J . Bid. Chem., 188, 1 (1951). Bechhold, H. von, and Hebler, F., Kolloid Z . , 31, 70 (1922). Callan, T. P., and Toennies, G., IND.ENG.CHEM.,ANAL.ED., 13,450 (1941).
Carbide and Carbon Chemicals Co., “Physical Properties of Synthetic Organic Chemicals,” 1950. Denis, W., and Reed, L., J . Biol. Chem., 71, 191 (1926). Horeischy, K., and Buhler, F., Mikrochemie oer. Mikrochim. Acta, 33, 231 (1947).
International Critical Tables, Vol. 5, pp. 30, 37, New York, SlcGraw-Hill Book Co., 1933. Kirsten, W., Abstracts 120th AM. CHEM.SOC. Meeting, p. 10B, 1951.
LahIer, V. K., Zbid, p. 11B; Chem. Eng. Sews, 30, 42 (1952). Medes, G., and Stavers, E., J . Lab. Clin. Med., 25,624 (1940). Nalefski, L. A., and Takano, F., Ibid., 36, 468 (1950). Ogg, C. L., Willits, C. O., and Cooper, F. J., ANAL. CHEU.,20, 83 (1948).
Sheen, R. T., Kahler, H. L., and Ross, E. M., IND.ENG.CHEM., ANAL.ED.,7,262 (1935). Siegfriedt, R. K.. Wiberley, T. S.,and Moore, R. IT.,ANAL. CHEM.,23, 1008 (1951).
Steyermark, A,, Bass, E., and Littman, B., Ibid.. 20, 587 (1948). Toennies G., and Gallant, D. L., J . Biol. Chem., 174,451 (1948). RECEIVEDfor review J u n e 12, 1952. Accepted September 20, 1952. Presented before the Dipision of Analytical Chemistry a t the 121st ,Meeting of the AMERICAXCHEMICAL SOCIETY,Buffalo, N. Y. Investigation supported in part by a research grant from the National Cancer Institute of the National Institutes of Health, United States Public Health Service a n d by a n institutional research grant of the .4mericitn Cancer Society.
