gain until 100% T was obtained with the broadened resonance lamp signal only. The original values of the atomic absorption were then obtained. See Figure 4. Errors begin to appear when nonatomic absorption is present at less than 30% T, Figure 5 . This discrepancy is related to the differences in the stray radiations from the two resonance lamps. The differences in stray radiations are subsequently minimized by introducing an interference filter in front of the photomultiplier. With this arrangement, 98% nonatomic absorption may now be corrected for without appreciable error. The recovered absorbance values of mercury standards in the presence of interfering substances were measured by adding to the mercury standards varying amounts of acetone giving nonatomic absorption down to 10% T level. The results are shown in Table 11. This instrument is designed initially for use in the rapid analysis of mercury in photographic raw materials. It has broader application in analyzing geological samples, such as soils and rocks, and provides a prospecting tool for mineral exploration. With appropriate application of chemistry, it is conceivable that analysis of mercury may be done by direct pyrolysis of the samples.
60
50
40
10
20
I
I
i
i
IO
20
30
1 40
50
---
1
I
100
ACKNOWLEDGMENT
%I M A A )
Figure 5. Corrected atomic absorption at various levels of non atomic absorption ulated by attenuating the signal from the sharp resonance lamp. Nonatomic absorption was obtained by introducing varying amounts of acetone vapor into the absorption cell. It was then observed that the signal from the sharp resonance lamp was less than the original adjusted value. The nonatomic absorption was then corrected for by adjusting the
I am grateful for the encouragement given by A. N. Ducksbury, 0. L. Goble, and N. B. Lewis. For the cooperation in the construction of the instrument, I thank G. Crickmore, E. Futschik, W. Lucas, D. San Miguel, and H. Wayne. Received for review March 1, 1968. Accepted July 1, 1968. Presented at the 6th Australian Spectroscopy Conference, Brisbane, Queensland, August 14-17, 1967.
Spectrophotometric Determination of Cysteine T. J. Bydalek and J. E. Poldoski Department of Chemistry, Uniaersity of Minnesota, Duluth, Minn. 55812
INa study (1) of the reaction between nitrilotriacetatoferrate (111) [Fe(III)NTA] and cysteine, a red-colored mixed complex was observed to form immediately on mixing and subsequently decompose. Investigation showed that the kinetics of the decomposition were complicated but the decomposition was determined to be caused by the reduction of nitrilotriacetatoferrate(II1) to an iron(I1) species by cysteine. This reduction is analogous to the reduction of Fe(II1) by cysteine which has been extensively studied (2). If the Fe(II1)NTA reduction was performed in the presence of 1,lo-phenanthroline (phen), any Fe(I1) formed was rapidly converted to the well-known, stable tris-1,lO-phenanthrolineiron(I1) complex [Fe(II)phenJ. In the absence of cysteine, the Fe(II1)NTA complex, however, reacted only very slowly with 1,lO-phenanthroline, and it appeared that the reaction could be used as a sensitive test for cysteine and possibly for thiols in general if
they possessed sufficient reducing capacity. This paper describes the application of the above reaction sequence to the quantitative determination of cysteine. EXPERIMENTAL
Reagents and Solutions. All reagents used were analytical reagent grade unless stated otherwise. An Fe(II1) solution was prepared by adding 20.0 ml of concentrated HC1 to 2.703 grams of FeC13 . 6 HzO (Baker) and diluting to 1 liter. The resulting pH was approximately 0.7. Standardization was made by titration with ethylenediaminetetraacetic acid (3). The solution was 1.003 f 0.002 x lO-*M in Fe(II1). A solution of nitrilotriacetate (NTA), approximately 1 to 3% in excess of the Fe(II1) solution, was prepared by dissolving 1.950 grams of nitrilotriacetic acid (Eastman) in 200 ml of distilled water containing 0.129 gram of NaOH (Baker) and diluting to 1 liter. The solution was 1.017 =k 0.003 x 10-2M. Standardization was made by titration of
(1) T. J. Bydalek, Unpublished Work, University of Wisconsin,
1965. (2) D. L. Leussing, J. P. Mislan, and R. J. Goll, J.Phys. Chem., 64, 1070 (1960).
1878
ANALYTICAL CHEMISTRY
(3) F. J. Welcher, “The Analytical Uses of Ethylenediaminetetraacetic Acid,” Van Nostrand, New York, 1958, pp 225-6.
Table I. Absorbance of Blank No. 1 2 3 4 5 6 7 8 9 10 11 12 13
14
Fe(II1) X lo4, M 1.003 1.003 1.003 1.003 1.003 3.009 5.015 6.018 6.018 6.018 7.021 6.018 6.018 10.03
NTA X lo4, M 1.017 1.017 1.017 1.017 1.017 3.051 5.085 6.102 6.102 6.102 7.119 6.102 10.17 10.17
Phen X 104,M 5.0 5.0 5.0 5.0 5.0 2.5 2.5
2.5 3.0 3.5 3.5 5.0 5.0 2.5
standard copper(I1) using murexide indicator ( 4 ) . The results agreed to 0.3% of the theoretical value and indicated that for the procedure given, this standardization was not necessary. A 5.0 X 10-3M solution of 1,lO-phenanthroline was prepared by dissolving 0.248 gram of 1,lo-phenanthroline monohydrate (G. F. Smith) in 250 ml of water. This solution was stored for no more than 3 days. The primary buffer used was a 2-amino-2-hydroxymethyl1,3-propanediol (Tris) (EastmankHCl mixture. Solutions of 1 M Tris and 1M HC1 were prepared and mixed as needed. Cysteine solutions were prepared by dissolving 0.0895 gram of cysteine hydrochloride monohydrate (Eastman) in distilled water and diluting to 500 ml. These solutions were used within 2 hours after preparation to avoid decomposition problems. A standard iron(II1) solution for the determination of the molar absorptivity of tris-1,lO-phenanthrolineiron(I1) was prepared by dissolving primary standard iron wire (0.5578 gram) in 20 ml of concentrated HCl and diluting to 1 liter. Apparatus. Spectral scans were made with a Bausch and Lomb 600 spectrophotometer equipped with a Sargent SRL recorder. All other absorbance measurements were made on a Beckman DU spectrophotometer using 1.00-cm cells. Solutions to be measured spectrophotometrically were prepared in borosilicate red glass (low actinic transmission) to avoid the reduction of Fe(II1)NTA by light. A Corning Model 10 pH meter with a glass electrode-calomel pair was used for all pH measurements after standardization with a buffer of pH 4.01 or 7.00. Procedure. The order and times of addition of the following recommended procedure are critical. A mixture of 10.0-ml of 1M Tris, 7.0 ml of 1M HCl (final pH 7.2-7.7) and 7.00 ml of 1OU2MNTA [1-3% excess with respect to Fe(III)] is pipetted into a 100-ml red glass volumetric flask. Fe(II1) (7.00 ml of l O - * M ) is then added and the walls of the flask are washed with distilled water. After allowing 2-3 minutes for the Fe(II1)NTA to form, 7.0 ml of 5.0 x 10d3Mphenanthroline are added, immediately followed by the addition of the cysteine sample, such that its concentration is in the range 10-5-10-4M. Ten minutes are allowed for complete color development, after which the sample is diluted to volume. Measurement is made at 510 nm within 30 minutes after the cysteine addition. The blank is treated identically except that no cysteine is added. The molar absorptivity of Fe(II)phena is determined in the pH range 7.2-7.7 using Tris buffer. An aliquot of the standard iron solution is pipetted (such that the final concentration (4) G. Numajiri, M. Kodama, and A. Shimizu, Nippon Kagaku Zusshi, 81,454 (1960); C. A., 55,15222e (1961).
PH 5.58 6.14 7.12 7.50 7.80 7.68
7.25 6.91-7.54 7.40 6.9-7.4 7.2-7.8 7.50 7.4-7.8 7.30
Absorbance 0.009 0.010
0.006 0.010
0.009 0.013 0.010 0.012 0.015 0.012 0.012 0.025 0.009 0.018
Max. rate abs units/30 min 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.002 0.002 0.002 0.015 0.013 0.010
is in the range 3-5 X 10-jM) into a 100-ml volumetric flask containing 10 ml of 1.OM Tris, 7 ml of 1.OM HC1 (final pH 7.2-7.7) and 1-3% excess NTA. To this mixture is added 7 ml of 5.0 x 10-3M phenanthroline and 1 ml of 10% hydroxylamine hydrochloride. Color development is complete in 10 minutes and the absorbance is measured in 1.00-cm cells at 510 nm. The NTA is added only to maintain conditions as close as possible to conditions encountered in the cysteine study. Identical molar absorptivities are obtained when NTA is absent, but 1-2 hours are required for complete color development. For purposes of comparison, the cysteine purity was determined by: (1) potentiometric titration ( 5 ) of known quantities of cysteine hydrochloride monohydrate with standard 0.1000M AgN03 using a silver wire indicator electrode, and (2) reaction of a known excess of 13-with known quantities of cysteine hydrochloride followed by a back-titration of the excess Is- with standard sodium thiosulfate (6). RESULTS
Cysteine Purity. Triplicate analysis by potentiometric titration gave the following values of the purity: 99.6, 99.7, and 99.8 %. The values obtained by the iodimetric titration procedure were: 99.5, 99.8, 100.2, and 100.5%. Assuming no determinate error in the two methods, an average (99.9 %) of all trials was used. The standard deviation was 0.35 %. Molar Absorptivity. The molar absorptivity of Fe(I1)phen3, calculated from Beer’s law, was 1.106 x 104M-1 cm-1 (average of five determinations in the range of 2-5 x 10-5M Fe) and agreed well with the literature values (7). REACTION OF FE(II1)NTA WITH 1,lO-PHENANTHROLINE Absence of Cysteine. The reaction of Fe(I1I)NTA with 1,IO-phenanthroline to produce tris-1,lO-phenanthrolineiron
(11) proceeds only very slowly in the absence of cysteine. Although the rate of reaction is slow, an initial absorbance (blank) was observed. The blank absorbance is caused by traces of Fe(1I)phena. All other components of the reaction mixture do not absorb at 510 nm. Preliminary studies indicated that the initial absorbance was higher, and the rate of reaction was increased, if the solutions were exposed to sunlight. Therefore, all solutions to be measured spectrophoto(5) S. Siggia, “Quantitative Organic Analysis via Functional Groups,” 3rd ed., Wiley, New York, 1963, p 571. (6) T. F. Lavine, J. Biol. Chem., 109, 141 (1935). (7) M. L. Moss, M. G. Mellon, and G. F. Smith, IND. ENG.CHEM., ANAL.ED., 14, 931 (1942). VOL. 40, NO. 12, OCTOBER 1968
1879
Table 11. Rate of Reaction in Presence of Cysteine Initial concentrations: F e w ) = 1,003 X lOP4M Phen = 5.0 X 10-4M NTA = 1.017 X 10-4M Cysteine = 1.0 X 10-5M
Per cent reaction at No. 1
Q
PH
3.70 2 4.30 3 7.12 4 7.35 5 7.44 6 7.53 7 7.71 8 7.84 9 8.23 No change with time.
2min
5min
25
...
...
30 89 86
84 83 85a 85a 80~ 620
10min 20min 29 36 33 44 93 95 88 91
458
metrically were prepared in borosilicate red glass (low actinic transmission) and this problem was avoided. Painting the flasks black was equally effective. The effects of pH, Fe(III)NTA, NTA, and phenanthroline concentrations on the blank reading were investigated and are given in Table I. The order of addition described in the procedure was followed except that no cysteine was added. The absorbance values given in Table I were obtained 2 minutes after the addition of phenanthroline and are the average of at least two runs. Where a pH range is given, at least six runs were made. It was observed that a stock solution of Fe(II1)NTA was stable for no more than 2 days, after which blank readings increased. Therefore, Fe(II1) and NTA were mixed as needed and 2 minutes were allowed for Fe(II1)NTA to form. Longer times for formation produced no change in blank readings. From Table I it can be seen that low blank readings and low increases in absorbance with time can be obtained under a variety of conditions (Nos. 1-11). As the FeOII), NTA, and phen concentrations are increased simultaneously, a much larger increase in absorbance with time is observed (Nos. 12-14), and these conditions are therefore not suitable for subsequent quantitative work. It should be pointed out that in the absence of NTA, very high absorbances due to tris1,lO-phenanthroline iron(I1) were observed. Presence of Cysteine. The overall reaction in the presence of cysteine can be written 2 Fe(II1)NTA
+ 2 Cysteine -I- 6 Phen 2 Fe(1IXphen)a
+ 2 NTA + Cystine
The visible spectrum clearly showed that Fe(1I)phen~was one product and 1 mole was produced per mole of cysteine initially present. The production of cystine was assumed by comparison with the reaction of iron(II1) and cysteine (2).
1880
No.
PH
1 2 3 4 5 6 7 8 9 10
7.68 7.68 7.50 7.51 7.20 7.20 7.50 7.50 7.70 7.70
ANALYTICAL CHEMISTRY
Preliminary studies were concerned with the quantitative and kinetic aspects of the reaction. Table I1 gives the values of per cent reaction at various times as a function of pH under the specified conditions. These values are the average of at least two determinations and are based on a cysteine purity of 99.9% and the determined molar absorptivity of Fe(I1)phen3. The order of addition used in the blank study was maintained and cysteine was added after the phenanthroline. From Table I1 it can be seen that the rate of reaction is relatively slow in the acid region, pH 3.7-4.3 (Nos. 1, 2). In the pH range 4.6-7.0, reproducibility could not be obtained and further investigations in this region were not performed. In the pH region 7.1-7.7, the reaction is relatively rapid but not quantitative under the specified conditions of Nos. 3-9. By increasing the Fe(II1) and NTA concentrations, but still maintaining them below the levels that lead to high blanks, the reaction can be forced to completion as seen in Table I11 (Nos. 5-10). All measurements were made within 10 to 15 minutes after the cysteine addition. Readings taken at 30 minutes showed no significant changes. At longer times, a tendency toward higher values (approximately 1-273 was observed. In all further work, the Fe(II1) and NTA concentrations were maintained at 7.02 x 10-4M and 7.12 X 10d4M, respectively. The pH was maintained in the region 7.2-7.7. Table IV gives the results of a cysteine variation over the concentration range of 10-5-10-4M. The values given are the average of 8 trials. Each cysteine concentration was obtained in duplicate after correction for the blank (duplicate) and the process was repeated four times. Interferences. The possible interference by amino acids, as well as inorganic anions, was investigated using a cysteine concentration of 7 x lO-5M. The substances were added to the system just prior to the cysteine addition. The following species showed no interference when present at 0.01M; glycine, serine, (DL) aspartic acid, (L+) glutamic acid, (L+) lysine. At a perchlorate ion concentration of O.lM, no interference was observed when measurement was made within 10 to 15 minutes after the cysteine addition, At concentrations as high as 0.4M perchlorate ion, no immediate interference was observed but, on standing, the perchlorate salt of tris-1 ,lophenanthroline iron(I1) tended to precipitate. Some species noticeably slowed down the rate of reaction. When measurement was made at 10 minutes after the cysteine addition, 0.01M histidine gave results 3% low; 0.01M tryptophan, 7% low, and 0.01M phosphate, 30% low. At a concentration of O.OOSM, histidine, tryptophan, and phosphate gave results that were low by 1, 6, and 16%, respectively. At both concentrations of these species, the absorbance increased to the maximum theoretical value if an additional 20 minutes were allowed for complete color formation.
Table 111. Per Cent Reaction as Function of Fe(1II) and NTA Fe(II1) X lo4, M NTA X lo4, M Phen x lo4, M Cysteine X los, M 3.01 3.01 6.02 6.02 7.02 7.02 7.02 7.02 7.02 7.02
3.05 3.05 6.10 6.10 7.12 7.12 7.12 7.12 7.12 7.12
2.50 2.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50
2.04 7.12 2.04 7.12 2.04 7.12 2.04 7.12 2.04 7.12
Reaction, 97 92 99 99 100 100 100 100 100 100
Table IV.
nature of the species and the color fading was not investigated.
Variation of Cysteine
Initial concentrations: Fe(II1) = 7.02 X 10-4M Phen = 3.5 X 10-4M NTA = 7.12 X 10-4M pH = 7.2-7.70
NO.
1 2 3 4 5 6
Cysteine found
Cysteine taken x 105, M
X 105, M Av of 8
1.018 2.036 3.054 5.090 7.126
1.02 2.04 3.06 5.06 7.12 10.2
IO. 18
detns
Absolute error x 105, M
+o.
002 +0.004 +0.006 -0.03 -0.006
$0.02
Std dev of 8 measurements x 105, M 0.02 0.01 0.02 0.03 0.05 0.05
Of the species tested, only tyrosine interfered seriously. At a concentration of O.OOSM, tyrosine gave results that were 3 x high. In the absence of phenanthroline and cysteine, tyrosine and nitrilotriacetatoferrate(II1) produce an orange-colored solution that absorbs at 510 nm. The species is not stable and the color fades after approximately 60 minutes. The
CONCLUSIONS
A rapid and simple spectrophotometric procedure for the determination of cysteine in the concentration range of lo-" 10-4M has been presented. Of the interferences studied, only tyrosine interfered to any appreciable extent. Studies are now underway to determine the general applicability of this reaction sequence to the determination of thiols and other reducing agents. The detailed reaction pathways have not been evaluated. A possible reaction sequence involves the labilization of the Fe(II1)NTA by reduction with cysteine to Fe(I1)NTA. This more labile species can then undergo ligand exchange to give the product, tris-1,lO-phenanthrolineiron(I1). However, it is also possible that the NTA is removed from Fe(II1) before or in the process of reduction with cysteine. Kinetic studies in these areas are needed. RECEIVED for review May 20,1968. Accepted June 14,1968. Work supported by the National Science Foundation, Grant GP-7319.
Quantitative Analysis of 1,2=PropanediolMonoesters of Long Chain Fatty Acids by Fluorine Magnetic Resonance Kazuo Konishi and Yoshiji Kanoh Industrial Research Laboratories, Kao Soap Co., Ltd., 1334 Minato-yakushubata, Wakayama-shi, Japan
THEI,~-PROPAP\'EDIOL monoesters of long chain fatty acids have been used as surface active agents to improve the foaming properties of edible oils. These 1,2-propanediol monoesters (1,2-rnonoesters) are usually prepared as a mixture of 1- and 2-monoesters accompanied with small amount of diester. Brandner reported that the equilibrium ratio of 1- to 2-monoester is dependent on the temperature ( I ) . A variation in the 1- and 2-equilibrium ratio may cause a difference in performances. Thus, it would be worth while to determine the equilibrium ratio as a necessary first step to establishing the relation between the equilibrium ratio and foaming properties. Recently, Manatt has reported the characterization of alcohols as their trifluoroacetates (TFA) by the fluorine magnetic resonance (2, 3). This method has certain obvious advantages; first, the chemical shifts of fluorine are in general larger than those of protons by about one order of magnitude. Second, one can observe the signals from three fluorine nuclei instead of one proton of an alcohol. Thus, at least, an increase of a signal to noise ratio of around 2.5 times should be realizable. In addition, because the observations are made on the fluorine spectrum, the signals from the protons in the molecule are far removed and the restrictions on the choice of solvent can essentially be eliminated. The 1,2-propanediol monoesters have one hydroxyl group (1) J. D. Brandner and R. L. Birkrneier, J. Am. Oil Chemists' Soc., 41, 367 (1964). ( 2 ) S. L. Manatt, J. Am. Chem. Soc., 88, 1323 (1966). (3) S. L. Manatt, ANAL.CHEM., 38, 1063 (1966).
in a molecule. The 1-monoester has a secondary hydroxyl group and 2-monoester a primary. For these types of hydroxyl groups, the present report describes an extension of Manatt's method (2, 3) to the quantitative analysis of 1,2monoesters of long chain fatty acids by the fluorine magnetic resonance. EXPERIMENTAL
Apparatus and Reagents. All fluorine spectra were obtained at 56.4 Mc. using a JNM-3H-60 type NMR spectrometer (Japan Electron Optics Laboratory Co., Ltd.) equipped with an integrator. The measurements were done at a temperature of 20 0.5 "Cand the spectra were recorded at a sweep rate of 1.69 cycle/sec. The chemical shifts between the signals were measured by the side band method using a standard frequency of 100 cycle/sec. A fraction collector SF-200 A type (Tayij kagaku-sangyo) was used for the elution chromatography of 1,2-monoesters. Analytical grade reagents of trifluoroacetic anhydride, chloroform, boric acid, acetone, and silicic acid, and commercial products of 1,Zpropanediol monostearates (1,2monostearates) were used. Trifluoroacetylation. About 200 mg of 1,2-monostearates and an excess (0.5 ml) of trifluoroacetic anhydride are mixed in a 10-ml conical beaker with ground stopper and allowed to stand for 1 hour at room temperature. After trifluoroacetylation, the beaker is transferred into a vacuum desiccator and the excess of trifluoroacetic anhydride and of trifluoroacetic acid formed by the reaction are removed at room temperature. The fluorine magnetic resonance spectra are measured with 20 solution of TFA derivatives in chloroform. VOL. 40, NO. 12, OCTOBER 1968
1881
