rapid and convenient dissolution technique, but when a large number of samples must be handled simultaneously the HFHN03method requires less operator time per sample. Precision and Sensitivity. The standard deviation of the spectrophotometric method was determined by carrying ten aliyuots, each containing 100 p g of uranium, through the extraction and spectrophotometric procedures. The relative standard deviation obtained was =t1 . 3 z . The practical molar absorptivity of uraniurn in the final solution was calculated to be 5.45 X 10'. This is lower than the practical
molar absorptivity found for uranium in the normal PADAP method, 6.59 X l o 4( I ) , but the sensitivity is still much higher than that given by dibenzoylmethane or thiocyanate. Limit of detection, calculated from the standard deviation of the blank, was equivalent to a U30, content of 15 ppm in an ore, using a 100-mg sample. RECEIVED for review August 18, 1969. Accepted October 27, 1969.
Gas Chromatographic Determination of SuHide, Sulfite, and Carbonate in Solidified Salts J. R. Birk, C. M. Larsen, and K. G . Wilbonm' Atomics International, A Division of North American Rockwell Corporation, P. 0. Box 309, Canoga Park, Calif. 91304 SEVERAL TECHNIQUES for the determination of sulfide and sulfite in the presence of one another have been suggested (f-5). For the most part these procedures are quite tedious and time consuming. In addition, erroneous results are caused by undesired alteration of the components during analysis and indirect measurement or measurement by difference. The molten carbonate process for the removal of sulfur dioxide from flue gases (6) has prompted the need for a rapid, sensitive, and reasonably accurate technique for the determination of minor quantities of sulfide and sulfite (1-20z) in the presence of large amounts of carbonate. A gas chromatographic technique was developed which depends upon the evolution of hydrogen sulfide, sulfur dioxide, and carbon dioxide from the sulfide, sulfite, and carbonate, respectively, when the sample is acidified. EXPERIMENTAL
Pure anhydrous sodium sulfide (analytical reagent) was obtained from Research Organic/Inorganic Chemical Co. This and all other chemicals which were also reagent grade were used without further purification. A Loenco Model 15A gas chromatograph in conjunction with a thermal conductivity detector was used for this work. The column of Poropak Q (Waters .4ssociates, Inc.) was 6 ft long and was operated at 100 "C with a helium flow rate of 60 cc/niin. The retention times for air, carbon dioxide, hydrogen sulfide, and sulfur dioxide were 40, 61, 155, and 316 seconds, respectively. Solidified samples and standards were obtained by drawing the fused salt mixture into 5-mm quartz tubes, which were subsequently broken after the melt solidified. The cylin1
Present address, Marquardt Corporation, Van Nuys, Calif.
(1) A. Kurtenacker and E. Goldbach, 2.Anorg. Allgem Chem., 166,
75,"H2S04-
/-------
/TO
ig I=
CHROMATOGRAPH\
I
1
REACTIONVESSEL
Figure 1. Reaction vessel and sampling system drical pieces of salt had a uniform composition and were more easily handled than powdered mixtures. The standard samples were prepared by combining the constituents-i.e., NazS or Na2S03with a eutectic mixture of Na2C03, K2C03, and Li,C03, mp 397 OC-in a quartz vessel and heating to a temperature of 425 "C in an inert atmosphere. Since the specific cation is unimpcrtant in the analysis, the combination of the alkali ions (K, Na, and Li) will be referred to hereafter in this paper as M-Le., MzC03, M2S03,and M2S. The procedure consisted of the following steps. The sample was weighed, placed in the reaction vessel shown in Figure 1, and the system evacuated. Value A was closed and 1.0 ml of a 7 5 z sulfuric acid solution was added to the sample. After dissolution the mixture was heated with a small hemispherical heating mantle at 200 "C for 1 minute. Value B was then closed, A was opened, and a 5.0-ml portion of the total volume (81.9 ml) was placed in the carrier-gas stream by means of a Perkin-Elmer sampling valve (Model 008-0659) and then analyzed.
177 (1927); C.A., 22, 362 (1928).
(2) J. H. Karchmer and J. W. Dunahoe, ANAL. CHEM.,20, 915
RESULTS AND DISCUSSION
(19.18). (3) P. Kivalo, ibid., 27, 1809 (1955). (4) R. Wollak, 2. A n d . Chem., 77, 401 (1929); C.A., 23, 4643 (1929). (5) I. M. Kolthoff and R. Belcher, "Volumetric Analysis," Vol. 111, pp 299-302, Interscience, New York, N. Y.,1957. ( 6 ) L. A. Heredy, D. E. McKenzie, and S. J. Yosim, U. S. Patent 3,438,722 (1969).
The selection of the most appropriate acid for the gas evolution reaction was of considerable importance. Ideally, the acid should be fast reacting witn the salt; unreactive with the evolved gases; nonvolatile to avoid interfering chromatographic peaks and equipment deterioration; and hygroscopic to avoid, during heating, the evolution of water which
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
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20
I
I
I
15 .
i 0
5 M Z S PRESENT
0
5
0
10
15
20
Figure 2. Instrument response as function of alkali sulfite present
Dashed line and triangles correspond to samples where sulfide content was greater than sulfite content Solid line and circles correspond to samples where sulfide content was less than sulfite content
Total weight of sample between 73 and 138 mg
M2S03 f MzC03 MzS03 MZC03 MzS0.9 MzC03 18.5’. 81.2b 18.8 f 0.5 81.2 f 0.5 MzS MzC03 MzS MzCOa MzS MzC03 12.9* 87.P 13.0 =t0.2 86.5 It 0.7 12.90 87. I C a Calibration made with pure anhydrous Na2S, Na2SOs, and Na2C03; five replicate analyses. b Determined by doing total sulfur analysis before and after evolution of the volatile sulfur compound. Carbonate determined by difference. c As weighed.
+
might condense on the sides of the reaction vessel and absorb the gases of interest or which could interfere with the sulfide determination because of overlapping of the water and hydrogen sulfide peaks. In the course of this work, several acids were tested and none were found to meet all these criteria. For example, perchloric acid reacted very slowly with the fused salt; 87 and 95z sulfuric acid reacted with the hydrogen sulfide to form elemental sulfur (Equations 1 and 2) : H2S HzS04 -.t S H20 H2S03 (1)
+
+
+
phosphoric acid reacted slowly and also interfered with the sulfur dioxide determination; and trichloroacetic acid was extremely slow in reacting. The acid which most closely met the requirements for this work was 75 sulfuric acid. The gas chromatograph and integrator response were initially calibrated using 42-1 88 mg of pure anhydrous salts. Approximately 100-mg samples of standard solidified binaryi.e., sulfide and carbonate; and sulfite and carbonate-salt mixtures were then analyzed. Table I indicates that excellent results were obtained. The procedure was then checked with mixtures of sulfide, sulfite, and carbonate which were made by rnixing various amounts of the two solidified binary standard mixtures with pieces of standard solidified carbonate. The analytical
z
274
y)
Figure 3. Instrument response as function of alkali sulfide present
M2S03 PRESENT (mg)
Table I. Analysis of Solidified Binary Standards Sample __ Per cent present Per cent foundo
10 (I
Table 11. Determination of Carbonate Present, mg Found, mg Recovery, 82.6 84.7 102.5 109.8 111.1 98.8 110.7 109.2 98.6 109.5 107.2 102.1 132.8 129.7 102.4 113.7 115.3 98.6 115 5 116.1 99.5 114.7 114.7 100.0 110.0 113.7 97.6 95.8 94.4 101.5 100.4 99.4 101 .o Average 100.2 Standard deviation 1.7z
results for sulfite (1-2073 and sulfide (1-lox)in the sulfite, sulfide, carbonate mixture are given in Figures 2 and 3, respectively. These plots show that there is a linear relationship between the amount present and the instrument response. The carbonate (80-95 %) in the sulfide, sulfite, and carbonate mixture was determined after calibrating the instrument with pure solidified carbonate. These results are presented in Table 11. The standard deviation for both the sulfide and sulfite was 6z and for the carbonate, it was 2z. Thus, reasonably accurate sulfide and sulfite results can be obtained using the procedure described herein along with the appropriate calibration curve such as Figures 2 and 3. The carbonate results, requiring only a single standard, were quite accurate. Total time for the determination of the three constituents was less than 6 minutes. The fact that the intercept in Figure 2 was less than zero demonstrates, particularly at the lower concentration levels, that the sulfite recovery was low while the opposite was true for the sulfide results (Figure 3). The low sulfite recovery was probably caused by sulfur dioxide absorption in the sulfuric acid. The reason for the apparently higher than theoretical sulfide recovery was less clear. Possibly the reaction between hydrogen sulfide and sulfuric acid (Equation 1) or hydrogen sulfide and sulfur dioxide (Equation 3)
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
is considerably more important at higher concentrations of sulfide than at lower concentrations. If this were the case, the lower results at the higher concentrations could cause the line to have an intercept greater than zero. Reaction 3 may also explain the results, as seen in Figure 3 (dashed line), where the sulfide was consistently lower in samples containing more sulfide than sulfite than in samples containing less sulfide than sulfite. Analyses of binary powdered mechanical mixtures (not previously melted) gave very poor results except in the case of mixtures of sulfide and carbonate. This is to be contrasted to the rather good results obtained with solidified samples. The problem resulted from the nonhomogeneity of the gas phase caused by evolution first of carbon dioxide or hydrogen sulfide followed by sulfur dioxide. This nonhomogeneity was demonstrated by the fact that the second of duplicate samples from the same gas mixture gave better results, and also, mixing
the gas in the reaction vessel yielded more accurate data. However, pelletizing the powdered samples between 25003500 lbs pressure gave results as good as those obtained with solidified samples. Pelletizing the powdered samples apparently allowed for a more uniform reaction resulting in a homogeneous gas mixture like that observed with solidified samples. This analytical approach appears to be generally applicable to a wide variety of anions-e.g., halides, acetate, nitrate, and nitrite as well as sulfide, sulfite and carbonate-which, in acidic solution, decompose or react to form gaseous products. The technique would be very valuable for rapid qualitative as well as quantitative analysis of many salt samples. RECEIVED for review September 15, 1969. Accepted November 17, 1969. Work upon which this publication is based was performed as part of Contract No. PH-86-67-128 with the U. S. Public Health Service, Department of Health, Education, and Welfare.
Nonaqueous Titrimetric Determination of Active Esters of Amino Acids and Peptide Derivatives Meir Wilchek, Mati Fridkin, and Avraham Patchornik Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel ACTIVEESTERS such as p-nitrophenyl esters (PNP), N-hydroxysuccinimide esters (NHS), or pentachlorophenyl esters (PCP), are widely used as coupling agents in the synthesis of linear and cyclic polypeptides (1-4). A simple and fast analytical procedure for their determination is therefore needed. PNP-esters are often determined spectrophotometrically by measuring the absorption of the p-nitrophenolate ion which is liberated on treatment of the esters with strong bases (5). The method is not of general use in the determination of other active esters. This paper describes a volumetric method for semimicro and microdetermination of active esters of amino acids and peptides (6). The method is based on the rapid and quantitative reaction between the active esters and the strong bases sodium methoxide and benzyltrimethylammonium hydroxide (Triton B). The reaction of active esters with sodium methoxide proceeds with one to one stoichiometry according to the following equation: RCOOR’ NaOCH3 + RCOOCHI NaOR‘ R = amino acid or peptide residue.
+
+
A solution of the active ester in anhydrous media (methanol, ethanol, dimethylformamide, aniline) is titrated with a solution of sodium methoxide. The end point in such a titration is readily detected by means of the indicator, thymol blue, which changes from yellow to blue in presence of a slight excess of sodium methoxide. The method is rapid simple, and accurate to within 2 to 3 %. The reaction of active esters with Triton B has one to two stoichiometry according to the equation: RCOOR‘
+ 2(CH&NCH2CsHsOH
-+
RCOO(CHJ3NCHzC6Hs
+
+
R’O(CH~)~NCH~C~H H20 S Titrations are carried out in pyridine or in dimethylformamide (DMF) and the end point is detected by means of the indicator ortho-nitroaniline. This indicator changes its color from yellow to orange-red in the presence of slight excess of Triton B. The titrations are rapid, simple, and accurate to within 2 to 4 %. PNP-esters cannot be determined accurately by this titration since the end points are not sharp. EXPERIMENTAL
(1) M.Bodanszky, Narure, 175, 685 (1955). (2) G.W.Anderson, J. E. Zimmerman, and F. M. Callahan, J . Amer. Chem. SOC.,85, 3039 (1963). (3) J. Kovacs, R. Ballina, and R. Rodin, Chem. Znd. (London) 1955 (1963). (4) J. Kovacs and A. Kappor, J. Amer. Chem. SOC.,87,118 (1965). (5) R. Schwyzer and P. Sieber, Helv. Chim. Acta, 40,624 (1957). (6) M. Wilchek and A. Patchornik, Bull. Res. Counc. Israel, 11.4, (1962).
Apparatus. Titrations were carried out using automatic burettes (5-10 ml volume) attached to 1-liter reservoirs equipped with drying tubes to protect the titrant from atmospheric contaminants. Reagents. Ethanol and methanol were of analytical grade. DMF, analytical grade, was dried over molecular sieves and distiiled at reduced pressure. Aniline was of analytical grade, freshly distilled at atmospheric pressure. All solvents must be tested for acidity and neutralized with sodium methoxide or with Triton B before titrations. Usually 1 drop of 0.1N sodium methoxide or of 0.05NTriton B was enough to produce a distinct color change of the
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