liter of calcium or a total of 7.49 meq. per liter were treated by the procedure. An average absorbance ( A , ) of 0.51 for calcium plus magnesium and an absorbance (Az) of 0.23 for magnesium was obtained. Dividing the total 7.49 meq. per liter by the absorbance 0.51 gave the value of K 1 equaling 14.7. Likewise K z was determined to be 10.4. Table I1 shows the data for the determination of the constants K 1 and K z and the average absorbance of the five samples from each vial. These constants were applied to five other blood serums. Three trials were made on each of the serums. I n every case the absorbances determined for the
three samples were nearly identical. Table 111 shows the observed absorbance and the calculated calcium and magnesium concentrations. Table IV compares the values found with the values given. After a day there were no significant changes in the readings. Per cent transmittance readings were converted to absorbances during the exrieriment. LITERATURE CITED
(1) Fabregas, R., Badrinas, A., Pruto, A,, Talanta 8, 804 (1961).
(2) Herrmann, R., Alkemade, C., “Chemical Analysis by Flame Photometry,” Wiley, Sew York, 1963.
(3) Jones, C. T., Ph.D. thesis, Oregon State University, Corvallis, Oregon, 1959. (4)Kirshen, N. A., M.S. thesis, Oregon State University, Corvallis, Oregon, 1961. (5) llartinez, B. F., Anal. Abstr. 6 , 3968 (1959). (6) Mason, W. B., ANAL. CHEM. 34, 23A-27A (1962). (7) Nakagawa, Genkichi, Wada, Hiroko, Tanaka, hIotohari, Talanta 10, 325, (1963). (8) Polyak, L. Ya., Ind. Lab. ( U S S R ) (English trans[.) 26, 809-13 (1961). (9) Sadek, F. S., Schmid, R. W., Reilley, C. S . , Talanta 2, 38-51 (1959). RECEIVED for review December 28, 1964. Accepted May 5, 1965.
Combined Infrared and X-Ray Spectrometric Method for Determining Sulfonate and Sulfate Concentration of Detergent Range Alkylbenzene Sulfonate Solutions S. D. KULLBOM, W K. POLLARD, and H. F. SMITH Research and Development Department, Continental Oil Co., Ponca City, Okla.
b We have developed a combined x-ray fluorescence and infrared spectrometric method for determining the alkylbenzene sulfonate and sodium sulfate concentration of alkylbenzene sulfonate-water slurries. Total sulfur content of the solution is determined by x-ray fluorescence measurements and the sulfonate content by infrared spectroscopy. The infrared sulfonate concentration is converted to per cent sulfur and subtracted from the total sulfur determined by x-ray fluorescence. This difference in sulfur is then expressed as per cent sodium sulfate in the original slurry. ITH THE INCREASING emphasis on w s p e c i a l structures and high quality of detergent range alkylbenzene sulfonates has come a new burden to the analytical chemist. S o t only must he meet stringent requirements of precision and accuracy in his analyses of plant production materials, but he must do this with increased speed. I n this laboratory we have attempted to replace some of the more tedious methods for sulfonate slurry analysis with newer, more rapid instrumental methods. We have previously reported ( 3 ) an infrared method for determining the sulfonate concentration of aqueous solutions of sodium and ammonium xylene sulfonates and a procedure for determining the sulfonate concentration of detergent range alkylbenzene sulfonates (4). This paper is a continua-
tion of the preceding reports and an extension of the previous methods, to include not only alkylbenzene sulfonate analysis, but also residual inorganic sulfates. T o accomplish both analyses, the techniques of infrared spectroscopy and x-ray fluorescence must be combined. Other instrumental methods for sulfonate concentration determinations ( I , 6) have been based on ultraviolet measurements and have a very severe limitation-i.e., all measurements are based on transitions of the pi-electrons of the aromatic ring. These transitions, and consequently the position and intensity of the absorption bands arising therefrom, are strongly influenced by molecular structure, especially changes in substitution pattern about the ring. Also, any nonsulfonated aromatic material which might he present will interfere with the ultraviolet methods. We developed the infrared method for sulfonate concentration determination because we felt that it would not suffer from this handicap. It was possible to devise an infrared method which is specific for the sulfonate group based on the absorption band arising from vibrations of the sulfonate group a t approximately 1172 cm.-l ( 2 , 5 ) . The classical and widely accepted methods for determining sulfonate concentration of aqueous sulfonate slurries involve a long and tedious gravimetric procedure or a somewhat better method involves complexation of the alkylbenzene sulfonate with p-toluidine
followed by several extractions of the aqueous solution with carbon tetrachloride. To the carbon tetrachloride extracts, one adds isopropanol and a titration indicator and titrates the isopropanol-carbon tetrachloride solution with sodium hydroxide and then back titrates with hydrochloric acid. The classical method for determining the residual sodium sulfate concentration of alkglbenzene sulfonate slurries consists of acidifying a solution of the alkylbenzene sulfonate with hydrochloric acid, extracting the resultant solution with several portions of diethyl ether, filtering the extracted aqueous solution to remove any solid material, and precipitating the remaining sulfate with barium chloride. The precipitated barium sulfate is then filtered, dried, weighed, and calculated as sodium sulfate. The time required for this rather tedious 1)rocedure is approximately eight hours. Consequently, any method to shorten the time and reduce the effort required for analysis would be of great benefit. EXPERIMENTAL
Materials (Infrared Method). Slurries of sodium alkylbenzene sulfonates were desalted and deoiled to obtain standards of combining weights ranging from 344 through 383. After the method was considered operational, a series of sulfonate slurries of unknown concentration was analyzed by both the infrared and chemical method and the results were VOL. 37, NO. 8, JULY 1965
1031
compared. Pure 2-phenylalkane sulfonates were studied to demonstrate the invariability of the 1172 cm.-l band molar extinction coefficient with the length of the aliphatic chain attached to the aromatic ring. For this work these compounds were analyzed gravimetrically and found to be 98 f 1% .pure. The impurities were determined to be water and residual sodium sulfate. Equipment (Infrared Method). A Perkin-Elmer Model 221G spectrophotometer was used. T h e sulfonate band system measured was in the region where the sodium chloride prism was the only effective dispersing element. A C I C type FT sandwich cell equipped with "IRTRAN-2" windows was used. The path length was approximately 0.025 mm. Working with aqueous solutions made it necessary to attenuate the reference beam of the instrument considerably. For this purpose a wire screen mounted in a demountable cell frame was used. The spectrometer was adjusted to operate under the following conditions: Recorder response, 2.5 seconds; speed
Table I. Concentration of Sulfonate Slurries as Determined by Infrared and Chemical Methods
Infrared analysis, wt. 70 sulfonate 45 46 46 45 45 44 44 44 45 45 44 44 45 45 46 45 41 41 47 47 37 46 43 44 44 45 44 43 49 49 48 47 41 42 42 43
46 43 42 43 42 45
1032
3 3 3 5 3 6 6 4 1 2 8 6 2 9 0 4 4 5 3 3 5 0 1 0 4 5 0 8 0 7 1 5
2 0 8 3 9 4 2 6 2 7
p-Toluidine titration, wt.
70
sulfonate 44.8 44.6 45.1 44.5 44.6 44.3 45.0 45.7 44.9 44.1 44.6 44.3 44.5 44.1 44.9 44.7 40.2 39.6 44.6 44.2 35.7 44.4 39.7 43.4 44.1 44.8 41.0 43.3 46.7 47.3 44.4 44.2 38.7 39.7 42.2 41.4 43.6 41.7 39.5 42.9 42.1 45.5
Difference
0.5 1.7 1.2 1.1 0.7 0.3 -0.4 -1.3 0.2 1.1 0.2 0.3 0.7 1.8 1.1 0.7 1.2 1.9 2.7 3.1 1.8 1.6 3.4 0.6 0.3 0.7 3.0 0.5 2.3 2.4 3.7 3.3 2.5 2.3 0.6 1.9 3.3 1.7 2.7 0.7 0.1 0.2 hlean error 1 .39 Rel. error + 3 . 2 %
+
ANALYTICAL CHEMISTRY
suppression, 0 ; abscissa scale, 25 cm.-l per cm.; scan speed, approximately 40 cm.-' per minute; normal gain, source current, 0.35 ampere, slit width at 1172 cm.-l, 262 microns. Equipment (X-Ray). The method for sulfate concentration based on sulfur analysis of the aqueous alkylbenzene sulfonate solution was developed using a Norelco Philips vacuum spectrometer with a tungsten target tube. The tube was operated at 50 kv. and 45 ma. A sodium chloride analyzing crystal was set a t 144.54' 20, offset 30". The sample chamber and optical path were purged with dry helium. Samples were counted for 100 seconds in duplicate.
Experimental Technique and Materials (X-Ray). T h e x-ray analyses were done on solutions prepared in the same manner as those for infrared analyses. I n fact, for the actual method development, the same standards and the same solutions made therefrom were used in both the infrared and x-ray calibrations. Since both analyses can be accomplished on approximately the same sulfonate concentration, it is necessary to make only one sample from the original slurry for analysis, thus eliminating any duplication of weighing and sample preparation. After the diluted sulfonate solution was prepared for analysis, the standard Philips x-ray fluorescence sample cell was equipped with a Mylar window and then filled with the sulfonate solution. The sample cell was then placed in the vacuum chamber of the spectrometer and radiation counts were taken immediately. These counts were used to interpolate the actual amount of sulfur present in the sample from a standard curve relating counts per second to sulfur concentration. Each analysis requires approximately five minutes of counting and calculation time. RESULTS A N D DISCUSSION
Infrared. The infrared method of analysis depends on the invariability of the absorption arising from vibration of the sulfonate group. The assumption was made in the beginning of this work t h a t variation in chain length or, for t h a t matter, even structure of the alkyl side chain on the aromatic ring would have a negligible effect on the position and intensity of the sulfonate band at 1172 cm-'. This assumption was verified by studies made on standard compounds- 2-phenylbutane, 2-phenyloctane, 2-phenyldecane, and 2-phenyltetradecane sodium sulfonate. Solutions of these compounds were made, and the molar absorptivity was determined for each compound. The average for all four was found to be 38.2 i 1.1 liters/mole-millimeter. This represents an average deviation of +2.9y0 relative. An earlier paper ( 3 ) on the xylene sulfonates demonstrated that changes in the position of alkyl substitution on the sulfonated aromatic
Table II. Effect of Sodium Sulfate Content on Activity Determination
Wt. SaeSOa based on bulk Sample sample
Absorbance of 5.17 wt.,%
active diluted sample
Dev. from av. of control absorbance
Av. 0.134 A
B
C
4.8
0.130 0.132 Av. 0.131 6.6 0.128 0.129 Av. 0.128 8.4 0.134 0.133 Av. 0.134
0.003 0.006
0.000
ring had little effect on the intensity and position of the sulfonate absorption band at 1172 cm.-l A series of typical plant production alkplbenzene sulfonates was desalted, deoiled, and taken as standards for this method development. The absorbance values for the sulfur-oxygen symmetrical stretching band of the dilute aqueous sulfonate solution were then related to concentration of sulfonate by the equation, Y = a b X , from which a = -0.0037 and b = 0.4279 X grams/ mole-millimeter. We next chose a large series of typical plant production samples which were being routinely analyzed by the chemical procedure and analyzed them by the infrared method. Table I shows the data obtained by infrared analysis compared to the data obtained by the chemical procedure. The mean error was found to be +1.39, assuming that the chemically derived values were correct. The relative error was +3.2%. We investigated the effect of certain parameters which could be expected to influence the infrared method of analysis. These effects were sodium sulfate concentration, sample aging, mixing time, and pH of the sample. Table I1 shows that there is no significant effect of Na2S04 on the sulfonate analysis. Table I11 shows that sample aging has little effect on the absorbance of the sulfonate stretching band a t 1172 cm.-l I t is apparent from these data that there is no significant difference in the absorbance measured five days after the solution was made compared to the absorbance on the first day. The effect of mixing time on the precision of analysis is shown in Table IV. The best mixing procedure involves use of an electric mixer on the aqueous
+
Table 111.
Av .
Effect of Sample Aging on Absorbance
Sample 1 Absorbance Dev. from av. 0.222 +o. 001 0.224 + O . 003 0.219 -0.102 0.221 0,000 0.220 -0,001 0.221 0.001
Day 1 2 3 4 5
sulfonate slurry. When using an electric mixer, sufficient mixing is attained after 1 minute. The effect of pH on the accuracy of the sulfonate concentration determination by infrared spectroscopy is shown in Table V. Data taken from aliquots of the same sample adjusted to pH levels ranging from 6 to 12.5 show no significant changes in the infrared value for sulfonate concentration.
Table IV.
Effect of Mixing Time
Absorbance of 5.1970 sulfonate solution 0.135 0.134 0.135 0.134 0.134 0.135 Av. 0.134
Mixing time, minutes
Table V.
PH 6 7 8
12.5
Effect of pH
70Active 49.2 49.4 49.3 48.8, 48.9
Table VI. Comparison of X-Ray and Chemical Sulfur Analysis of Sulfonate Slurries
Wt. 70 s (x-ray) 5.07 4.98 5.09 4.95 5.06 5.00
5.02 4.86 4.77 4.83 4.74 4.81 4.78 4.81 4.87 4.76 4.64 4.57
Wt. 70 S (Chem.) Difference 4.81 0.26 4.59 0.39 5.03 0.06 4.90 0.05 5.05 0.01 4.90 0.10 4.79 0.23 4.85 0.01 4.90 -0.13 4.79 0.04 4.87 -0.13 4.94 -0.13 4.77 0.01 -0.06 4.87 4.90 -0.03 -0.06 4.82 -0.07 4.71 -0.25 4.82 Mean error +0.017 Rel. error 0 . 35y0
Sample 2 Absorbance Dev. from av. 0.103 -0,001 0.103 -0.001 0.104 0.000 0.105 +0.001 -0.001 0.103 0.104 -0.001
Although the infrared method for sulfonate concentration determination looked quite good at this point, we had yet to show the feasibility of the x-ray procedure for determining residual sodium sulfate concentration in the sulfonate slurry. This determination depends not only on the accurate analysis of the total sulfur present in the sulfonate slurry by x-ray fluorescence, but also on accurate sulfonate determination by infrared spectroscopy. Typical plant production alkylbenzene sulfonates were analyzed for us by a chemical procedure and then by x-ray fluorescence. A comparison of the weight per cent sulfur found in a series of 18 alkylbenzene sulfonate slurries by x-ray fluorescence and chemical analysis is shown in Table VI. The agreement between the two methods is excellent. These 18 samples were analyzed for sulfonate content by infrared spectroscopy and the residual sodium sulfate content was calculated in the following manner. From a known aqueous dilution of the sulfonate, a 100-second radiation count was made. The count rate of the diluted sample was compared to a calibration curve established using known sulfonate concentration as standards. The per cent sulfur in the solution times the dilution factor gave the total sulfur concentration of the sulfonate. An “active” sulfur is calculated from the activity and the molecular weight of the sulfonate which has been determined independently. The difference between “total” sulfur and “active” sulfur represents sulfur due to inorganic sulfate. The inorganic sulfur times the appropriate factor gives per cent sodium sulfate in the sulfonate. The results of these calculations are shown in Table VII, in which we have listed the weight per cent sodium sulfate as calculated using x-ray and infrared data. I n the second column are listed the sodium sulfate data obtained by barium precipitation, the classical method for determining residual sodium sulfate. The mean error, assuming the chemically determined wlfate concentration is correct, mas found to be +0.016. The relative error is 0.44Yc.
Table VI11 shows data obtained by the combined infrared and x-ray methods for sodium sulfate concentration compared to that obtained by chemically analyzing the sample for total sulfur content and determining the amount of sulfonate by chemical means, expressing the sulfonate as sulfur and subtracting this value from total sulfur and expressing the difference in sulfur as sodium sulfate. The mean error between these two analyses, if we consider the chemical procedures to be correct, is -0.20. The relative error is -5.0%.
Table VII. Comparison of Na&04 Content as Determined by Instrumental Analysis and by Ba Precipitation
Wt. 70 Xa2S04 4.5 3.7 4.2 3 9 4.4 4.4 4.5 3.9 3.2 3.5 3.2 3.6 3.2 3 1 3.3 3 1 4 1 3 8 1 4
Wt. yo Na2S04, Ba precipitation 3.6 3.7 4.1 4 2 3.2 3.4
Difference 0. 9_
0.0
0.1
-- 0 . 3
1.2 1.0 nR 0.6 -0.5
3.6
3.3
3.7 3.5 0.0 3.6 -0.4 3.9 -n . 3_ 3.7 -0 5 3 5 -0.4 4 1 -0 8 40 -0 9 40 0 1 4 3 -0 5 1 3 0 1 hlean error +O 016 Rel. error 0 447,
Table VIII. Comparison of Na2SOa Content as Determined by Instrumental Analysis and by Indirect Chemical Analysis
Wt. %
Wt,.
70
Na2SOr 4.5 3.7 4.2 3.9 4.4 4.4 4.5 3.9 3.2 3.5 3.2 3.6 3.2 3.1 3.3 3.1 4.1 3.8
I\Ja2S0,, chemical analysis 3 5 2 6 4 4 4 0
Difference
4 6
4 3 3 3 3 3 4 3 4 3 3 4 5
1 0
1 1
-0 2 -0 1
-0 2
2 0 1 4 3 0 -0 9 7 -0 8 -0 3 -0 5 -0 -1 1 9 -0 6 -0 9 -0 6 -1 hlean error -0 Re1 error - 5
VOL. 37, NO. 8, JULY 1965
2
1
6 7
2
6 7
3 0 6 5 8 8
20 O cc 1033
I n summary, we have successfully combined x-ray fluorescence spectrometry and infrared spectroscopy to effectively shorten the analysis time required for determining sulfonate and sulfate concentration of neutralized alkylbenzene sulfonate slurries. Both analyses can be completed in less than thirty minutes. This is in contrast to approximately thirty minutes required for the determination of sulfonate concentration by the chemical method and approximately eight hours elapsed
time required for residual sodium sulfate concentration determination by the classical chemical procedure. This saving of time is substantial and can be achieved with no loss in precision and accuracy compared to the presently acceptable chemical methods. LITERATURE CITED
(1) American Society for Testing Ma-
terials. PhiladelDhia. Pennsvlvania. "ASThI Standar'ds," Part io, pp: 1045-8, 1961.
(2) Barnard, D., Fabian, J. M., Koch, H. P., J . Chem. SOC.,1949, 2442. (3) Kullbom, S. D., Smith, H. F., ANAL. CHEM.35, 912 (1963). ( 4 ) Kullbom, S.D., Smith, H. F., Paper Yo. 212, 14th Pittsburgh Conference on Analytical Chemistry, and Applied Spectroscopy, March 8, 1963. (5) Schreiber, K. C., ANAL. CHEM.2 1 , 1168 (1949). (6) Weber, J. W., Jr., Morrisj J. C., Stumm, Werner, Ibid., 34, 1844 (1962). RECEIVED for review February 19, 1965. Accepted April 29, 1965.
Colorimetric Quantitative Method for Determining Glycine in Presence of Other Amino Acids J. P. JEWELL, MARY J. MORRIS,' and R. L. SUBLETT Department o f Chemistry, Tennessee Technological University, Cookeville, Tenn.
b Color reaction between glycine, ethyl chloroformate, and pyridine was studied as a quantitative colorimetric determination of glycine. The method could b e used for the determination of glycine in the presence of other amino acids and in acidic hydrolyzates. The results are quantitatively the same for glycine, glycine hydrochloride, and sodium glycinate. The method is simple and does not require expensive equipment.
A
EARLIER PUBLICATION (2) from this laboratory described a specific colorimetric qualitative test for glycine using the reaction of glycine with ethyl chloroformate and pyridine. It was stated in that publication that work was in progress to adapt the reaction to a quantitative method. LVhile this work was in progress, Umberger and Fiorese (3) reported a quantitative colorimetric procedure using pyridine and p-nitrobenzoyl chloride. In their report no data were given for the analysis of amino acid mixtures and protein hydrolyzates. Also, this laboratory found that in the presence of water most amino acids give a purple-blue color with pyridine and p nitrobenzoyl chloride, the intensity of which varies with the amount of water present. Other colorimetric methods for the determination of glycine are described in the literature ( I ) . However, they require a preliminary separation of glycine from other amino acids. The present investigation concerns the determination of glycine in amino acid mistures and protein hydrolyzates, and involves the color reaction of glycine
N
1 Present address, T-anderbilt Hospital, Sashville, Tenn.
1034
ANALYTICAL CHEMISTRY
with ethyl chloroformate and pyridine. The method does not require separation of glycine from other amino acids prior to testing and the color reaction is quantitatively the same for glycine, glycine hydrochloride, and sodium glycinate. The method is simple and does not require expensive equipment. EXPERIMENTAL
Reagents and Apparatus. T h e reagents used were ethyl chloroformate (Distillation Products Industries, White Label), pyridine (Matheson Coleman & Bell, Spectroquality, dried over sodium hydroxide) , n-butyl alcohol (redistilled) , amino acids (Distillation Products Industries, White Label), and proteins (Nutritional Biochemicals Corp.). Apparatus used, other than standard laboratory glassware, were a Bausch and Lomb Spectronic 20 colorimeter, a microburet (Beckton Dickinson and Co., B-D Yale syringe microburet Model Yo. l l l ) , and graduated centrifuge tubes. Procedure for Calibration Curve. T h e procedure for t h e development and meawrement of the color produced when glycine is reacted with ethyl chloroformate and pyridine is outlined below in successive steps. With a microburet, a sample of a standard aqueous glycine solution (0.0050M) was measured into a 15-ml. graduated centrifuge tube. The sample was dried a t 125' to 130' C. for 2 to 3 hours. The dried sample was thoroughly mixed with approximately 0.1 ml. of pyridine by alternately heating in a boiling water bath and stirring with a glass stirring rod. Three portions of ethyl chloroformate (a total of 0.5 ml.) were added dropwise with thorough mixing after each addition. The temperature was maintained a t about 100" C. throughout these additions. Heat-
ing and stirring were continued after the last addition of ethyl chloroformate for approximately 1 minute, or until a red color of maximum intensity was observed. Immediately after removal of the sample from the constant temperature bath the sample was aspirated, because excess ethyl chloroformate produces a green coloration. The sample was immediately diluted to 10 ml. with butyl alcohol and its absorbance was determined at 420 mp, the wavelength of maximum absorbance. Redistilled butyl alcohol was used as the reference for zero absorbance. Organic solvents such as those listed in a previous article (2) could be used. The absorbance of known samples (ranging from 0.081 ml. to 0.405 ml.) of the standard aqueous solution of glycine was determined by the above procedure. Data are given in Table I and were used to obtain a Beer's law curve. I n the procedure, sodium glycinate or glycine hydrochloride can be substituted for glycine in equimolar concentrations to obtain identical calibration curves. Determination of Glycine in Presence of Other Amino Acids. h study was made of the effect of other amino acids on the above procedure. The absorbance of glycine was compared with t h a t of a mixture containing equimolar amounts of glycine and the following amino acids : DL-a-alanine, p-alanine, L(+)glutamic acid, L ( -) leucine, DL-isoleucine, DL-lysine monohydrochloride, DL-p-phenylalanine, and DL-threonine. The preparation and determination of the absorbance is outlined above. The results showed that the absorbance of the glycine samples containing other amino acids averaged 0.07 higher than the samples containing only glycine. Samples containing only threonine in the same concentration as above showed a n average absorbance of 0.05.
