heated sufficiently so that low results were obtained. Early experiment,ttion using zinc oxide for phosphate removal indicated t,hat, heating a t approximately 850” C. is necessary under the above conditions for complete decompcisition of any zinc sulfate formed during the combustion. 153th the surface area of zinc oxide powder in the positions indicated under Procedure, no phosphorus oxides are left loosely bound to the silica of the inner combustion tube walls. Thus, after removal of the combustion tube packing, the combuslion tube may be washed with water for sulfuric acid removal. Further work with .:he use of the zinc oxide reagent could indicate the route of phosphorus and its oxides in other combustion procedures such as the carbon and hydrogen determination.
ACKNOWLEDGMENT
Special appreciation is expressed by the authors to Herbert S. Aaron of these laboratories for supplying the pure 0-alkyl alkylphosphonothioic acid salts used in testing this separation method. LITERATURE CITED
(1) Colson, A. F., Analyst 88,26 (1963). ( 2 ) Corliss, J., U. S. Dept. Comm., Office Tech. Serv., Report AD 278,160
(1962). (3) “Czechoslovak Fine Chemical Standards,” Vol. 11, p. 412, Chemapol, Praha 3, Czechoslovakia, 1957. ( 4 ) Ellison, M., AnaLyst87, 389 (1962). ( 5 ) Fritz, I., Yamamura, S., ANAL. CHEM.27. 1461 i1955). (6) Goulden, R . , “Comprehensive Analytical Chemistry,” C. L. and D. W. Wilson, Eds., Vol. lB, p. 420f. Elsevier, New York, 1960. ( 7 ) Hallett, L. T , Kuipers, J. W.,IND. ENG.CHEht., AXAL.ED. 12, 357 (1940).
(8) Hodgman, C. D., ed., “Handbook of Chemistry and Physics,” 38th ed., Chemical Rubber Publishing C o. Cleveland, 195G57. (9) Kanzelmyer, J. H., “Treatise on Analytical Chemistry,” I. M. Kolthoff and110, P. J. Elvine. eds.. Part IT. Vol. p. Interscicice, New YoFi: i G i .3. ~
-I
(10) Kar, K. P., Xath, S . , J . Sei. 2nd. Res. (India)16B, 563 (1957). (11) hleier, E., Mikrochim. Acta 1961,70. (12) Schoniger, JY., Ibid., 1955, 123. (13) Schiiniger, IT.,Ibzd., 1956,869. (14) Smith, W. H., A N A L .CHEY.30, 149 (19581. \ - - - - ,
(15) Steyermark, A . , “Quantitative Organic Microanalysis,” 2nd ed., p. 295f, Academic Press, Yew York, 1961. (16) Zimmerman, R., AWzkrochem.,Wikrochim. Acta 31, 13 (1943).
~
RECEIVEDfor review July 5, 1963. Accepted October 10, 1963. Division of Analytical Chemistry, 145th Meeting, ACS, New York, September 1963.
Spectrophotometric Micro and Submicro Determination of Sulfur in Organic Substances with Barium Chloranilate PIERRE STOFFYN and WILLIAM KEANE Mclean Hospital Reseclrch laboratory, Belmont, Mass.
b A method is described for the spectrophotometric determination of sulfur in organic substances in the range of 0.3 to 100 rug. of sulfur in samples weighing up to 3 mg. dry weight. Relative error is *270 in the 1-119. range and + 1 % in the 20-pg. range, The samples are mineralized according to Carius bsy oxidation with nitric acid or aqua regia on a microscale, and the resulting sulfate is determined with barium chloranilate. Interfering cations are eliminated by exchange in the presence of Dowex 50 in the ammonium form.
T
HE QUANTITATIVE DETERMINATION
of small amounts of sulfur in organic substances or in complex mixtures like sulfur-containing lipid extracts of tissue, was attempted by the Lysyj and Zarembo method ( 4 ) . This procedure combines the very rapid oxygen-flask combustion method of Schaniger ( 6 ) with spctrophotometric determination of resuli,ing sulfate with barium chloranilate. This last procedure originated by 13ertolanici and Barney ( I ) , was adapted by Lloyd (3) for the analysis of small amounts of inorganic sulfate in inaterial of biological origin and further scaled down
MUSS., and Department o f Biological Chemistry, Harvard Medical School, Boston;
by Spencer (6). It is rapid, sensitive, and compatible with the presence of various anions including phosphate. Interfering cations are eliminated by ion-exchange. Sulfur can be determined over a a i d e range by the method because the absorbance of the final solution containing acid-chloranilate ions can be read either a t 530 mp or, with a very large increase in sensitivity, a t 32i.5 mp. Inaccurate results were obtained however, in the analysis of very small amounts of sulfur when the combustion of the sample was performed in the oxygen flaqk. Therefore, the Carius procedure of mineralization was used. It was found to be simple and safe when scaled down as described, and led to good results. Moreover, a large number of combustions can be performed a t the same time.
EXPERIMENTAL
Reagents. Double-distilled water is used throughout the procedure. OXIDIZJNG AIIXTURI:. Sitric acid. $1). gr. 1.42 A . R., containing 2 mg. of NaCl per nil., or aqua regia, freshly prepared mixture of 3 volumes of nitric acid and 1 volume of concentrated HCI
containing 2 mg. YaC1 per ml. of the mixture. BUFFER. Stock solutions: (A) 6.3 grams of ammonium formate dissolved in 50% aqueous ethanol to make 100 nil. and (B)4.2 ml. of formic acid, sp. gr. 1.2, diluted to 100 ml. with 50% ethanol. For use, a mixture of 1 volume of =\ and 4 volumes of R is diluted 20 times with 507, ethanol. The apparent pH is 4.4. I O N EXCH.4XGER. Dowes 50 X-8, 50 to 100 mesh, is preconditioned by washing successively, and twice repeatediy, with I S HC1, water, I S XII,OH, and water. The resin is finally washed with, and then kept in 507, ethanol. After long periods of storage, the resin is washed a few times by decantation with 50% ethanol before use. BARIUhl CHLORAKILATE. Prepare from chloranilic acid and BaCI? ( I ) or obtain from commercial sources. For use, a suspension of 100 mg. of barium chloranilate in 10 ml. of buffer solution is shaken for I O minutes. This suspension can be kept for several days. STANDARDS.Standard solutions of inorganic sulfate arc prepared from anhydrous potassium sulfate dried a t 110” C. under vacuum. Solutions containing known amounts of organic sulfur, are prepared from methyl GOp-toluylsulfonyl- a - D-galactopyranoside in pyridine, of ethyl 2,3-isopropyliciene-4O-p-toluylsiilfo~i~~l-~-~-lyxopyranoSe in VOL. 36, NO. 2, FEBRUARY 1964
a
397
Table 1.
Recovery of Inorganic Sulfur from KzS04
S. ue.
Calculated 12.5 25.0 50.0
0.25 0.50 1 .oo
1.84 2.00 3.00 3.62
Found Individual determinations, 530 mp 12.5; 12.5; 12.5; 12.0 25.0: 25.0: 25.0: 25.0: 24.8: 25:3 50.0; 50.0; 50.0; 50.0; 49.7; 50.2; 49.0 Individual determinations, 327.5 mp 0.25; 0.2.5; 0.25; 0.28 0.50; 0.50; 0.46; 0 . 4 6 1.00; 1.01; 1.03; 1.06; 0.98; 0.98; 0.98; 0.97 1 . 8 8 ; 1.83; 1.77 1.98; 1.98; 1.98; 1 . 9 3 3.00; 3.00; 3.00; 3.03; 3.70; 3.56; 3.63
chloroform and of methionine in 50% ethanol. ADDaratUS. 411 elassware is cleaned witg *nitric acid, rynsed with distilled water, and finally with double-distilled water. Polyethylene stoppers are rinsed with water, shaken in a mixture of chloroforni and methanol (2: 1) and dried in the air. Micro Carius tubes are made in pairs from prewashed 20cm. lengths of borosilicate glass tubing 4-mm. o.d. with walls 0.8 mm. thick. The middle part of the tube is heated in a small torch flame, and by pulling and separating in the flame two tubes are obtained with sealed tips about 6 mm. long and tapering to about 1 mm. in diameter. By further heating of the extremity of the tip, a glass bead 2 mm. in diameter is formed at the end. After filling, the open end of these tubes is sealed to form a 20 to 25 mm. long and
~~
Table
II.
Recovery of Sulfur from Organic Substances
Substance Methionine Methyl G-o-p-toluylsulfonyl6-D-galactoside
S,Pg. Found CalIndividual determinations. 530 mp ' calculated 23.9; 23.9; 23.0; 23 4 23 9 25.1
1.58 2.39
Methionine Methionine
Methyl G-O-p-toluylsulfonyl-@- 2.51 D-galactoside
Table 111.
25.4; 25.4; 25.4 Individual determinations, 327.5 m p 1.56; 1.67; 1 . 6 3 ; 1.64 2.25; 2.37; 2.50; 2.51; 2.40; 2.40 2.52; 2.53; 2.57; 2.46
Mean 23.7
Recovery.
%
"
I
99.2
25.4
101.2
Mean 1.62 2.42
102.5 '101.3
2.52
100.4
Recovery of Sulfur from Methionine Added to Glucose
S. uei Found Individual determinations, Substance Calculated 327.5 mp 2.18 2.03; 2.10; 2.17; 2.18 Methionine 2.18 2.19; 2.10; 2.25; 2.22 Methionine 3 mg. glucose
+
Table IV.
72
97.3 100.4
Recovery of Sulfur from Methionine Added to Sulfur-Containing Lipid Extracts
s, r g .
Substance Lipid extract containing 1.3y0P Sulfatides preparation
Lipid extract Sulfatides preparation
398
Recovery, Mean 2.12 2.19
__
Recovered ReContained Individual deterIndividiial deter- covery, minations, 530 mp Added minations, 530 mp 7c Mean Mean 3 6; 3 . 6 ; 3 . 7 23.9 27 6; 27.6; 27.8 100.8 3.9 28.1 20.8; 2 1 . 2 ; 21.0 23.9 45.5; 45,6; 45.7 101.8 21.0 46.0 Individual determinations, 327.5 m p Mean 1.09; 1.19; 1.12 1.10; 1.09 3.03; 3 . 1 1 ; 3.10 3.14; 3.12
ANALYTICAL CHEMISTRY
Individual determinations, 327.5 m p Mean 2.39 3.27; 3.38; 3.40 96.9 3.50; 3.46 2.39 5.48; 5 , 6 3 ; 5 65 103.0 5.85
1-mm.-thick tip with very thin capillary lumen. During the combustion, these tubes are placed with the beaded tip up into clean glass test tubes 10 em. long and 6-mm. i.d., which are themselves contained in shields made of copper tubing 11 em. long and 10-mm. i.d. and closed at, one end by flattening in a vise over a length of 1 ern. The remainder of the procedure is carrkd out in borosilicat'e test tubes 85 mm. long and 15-mm. o.d. with standard ground necks S o . 9. Corresponding polyet'hylene stoppers are used for leakproof closure. Procedure. Solid samples are weighed in tared combustion tubes on a microbalance. Samples in solution are introduced into the combustion tubes with a calibrated micropipet, and 25 pl. of pure solvent is added afterwards to rinse the walls of the tube. Solutions are evaporated to dryness by putting the tubes vertically inside a 250-rnl. filtration flask stoppered with rubber, and then lowering .the pressure slowly with a water aspirator. Once t,he vacuum is established, the evaporation can be accelerated by partly immersing the filtration flask in lukewarm water. After drying, 50 pl. of osidizing mixture is int,roduced into the tube, which is then sealed and heated in an oven a t 270" t,o 280" C. for 8 hours. -Aft,er the combustion, the tubes are cooled in their 'shields to room temperature, then cooled by immersion in a solid COz-alcohol mixture where their content' freezes. The tubes are t,hen withdrawn one by one from their shields and the beaded tip is heated carefully for a few seconds in a small flame in order t,o dislodge any liquid from t8hatt,ip. The tubes are put back in the cooling mixture in an upright position for a short time. To open a combustion tube, a scratch is made with a glass scorer just under the bead which is then very e a d y broken off with pliers. The opened tubei; are left, standing until they reach room temperature. T o transfer the content of the combustion tubes into test tubes, a scratch is made with a glass scorer about 2 mm. from the extremity of the capillary tip which is then introduced inside a test tube, and, by pressing against' the inside wall of the test tube, the bip is broken at the scratch. The content, of the combustion tube is transferred quantitatively int'o the test tube by sloivly forcing 0.5 ml. of water through tjhe combustion tube with a syringe. The content of the test tubes is evaporated to dryness a t room temperature overnight in a vacuum desiccntor containing CaC12 at the bottom and a cup of S a O H pellets. T o the dry residues are added 1.2 ml. of 50% ethanol and 7 drops (about 0.1 ml.) of the suspension of Dowes 50 in t.he ammonium form in 50y0ethanol. This is done by aspirating the Dowes suspension in a pipet' and holding the pipet, in a vertical position allowing t,he resin beads to settle in a uniform bed which is then delivered as a liquid. The test' tubes are stoppered
however, blanks and standards have to be handled through the entire procedure including the combustion step because higher results were obtained in certain cases after prolonged heating of blanks, standards, or samples with aqua regia a t 2iOo C. as compared to nbncombusted blanks and standards or to sample? combusted for shorter periods of time.
2or
.I0
t 650
600
550
500
450
400
W A V E iE N G T U
Figure 1 .
350
300
250
lmrl
Adsorption spectra
_ _ _ _o f
a solution resultirlg from the addilian of 1 0 0 0 pg. of phosphorus (as NH~H~POI)to a barium chloranilate blank Firepored as described A similar blank was used far in the procedure. reference in the spectropholameter. A maximum is observed at 3 2 5 mjr Obtained when 4 psi. of sulfur (as sulfate) was a d d e d to an identical blank. The maximum i s observed at 3 15 mp
with polyethylene st'oppers and shaken in a slanted position on a shaking machine in s w h a wav that the resin is w l l swirled with the liquid without, however, reaching the stopper. .Inumber of tubes are held conveniently in a ivire rack b!. means of a cloth band lined with a plastic foan- pad. After I 5 minutes, the content of the tubes is centrifuged a t about 3000 r.11.m. for 30 seconds. One niilliliter of the supernatant solutions is pipetted ir to test tubes containing 1 ml. of the bctrium cliloranilate suspension. .ifter closing with polyethylene stoppers, the tubes are shaken as described in the previous step for 15 minutes, and centrifug;ed for 30 seconds a t 3000 r.11.m. As c8mall particles of barium chloranilate t m d to adhere to the walls of the tubes, the centrifugation is interrupted at thE,t stage and the tubes are inverted for a second or two in such a way that the supernatant solution washes the walls of the tube while the precipitate remains a t the bottom. .1 second centrifugation is run for 30 seconds. The supernatant solution is transferred into 1-ml. quartz cuvettes and the absorbance is read on a Beckman DLspectrophotometer a t 327.5 mp tor amounts of sulfur srrdler than 4 pg. and a i 530 mp for 20 tcl 100 p g . of sulfur. Sulfur amounts are computed from a st,andard curve. Conditions difficult 1 o control such as the variation in seniitivity resulting from the use of difhrent batches of barium chloranilate make it necessary to prepare a standard cwve with known amounts of inorganic sulfate for each set of determinations. For determinations in the 20- to 100-pg. range, blanks and standards do not need to be handled through the combustion part of the procedure but are simply pipetted into t,est tubes with a micropipet, 50 p l . of combustion mixturt> and 0.5 ml. of water are added, and the mixture is evaporated in a desimitor and then further processed as described. 13lanks arc prepared in the same way by evaporation in the te
