Extraction and Flame Photometric Determination of Iron OSCAR MENISI and T. C. RAINS Analytical Chemistry Division, Ook Ridge National laboratory, Oak Ridge, Tenn.
b A flame photometric procedure for the determination of iron in a variety of materials including thorium, uranium, fluoride, and phosphate is described. The iron i s extracted with 4-methyl-2-pentanone from a 6M hydrochloric acid medium, after which the emissivity of the iron in the organic phase is measured at a wave length of 372 mp. The emissivity of iron atomic lines is 20 times greater in a 4-methyl2-pentanone than in an aqueous medium, while that of FeO bands is increased approximately only tenfold. Iron can be quantitatively extracted with 4-methyl-2-pentanone from 4 to 8M hydrochloric acid solutions with an equilibration period of 1 minute. Emulsion formation can b e prevented by control of speed and duration of shaking. Of 15 elements tested for interference, only chromium (VI), molybdenum, and tin interfere when they are present at concentrations greater than 150 times that of the iron. Solutions of 1M fluoride, phosphate, and sulfate do not interfere, but 0.1M soiutions of nitrate or perchlorate interfere b y quenching the emissivity of iron. The calibration curves are linear for the concentrations which were tested-namely, within the range of 1 to 50 pg. per ml. The coefficient of variation of the method, depending on the material, ranges from 2 to 4%.
T
DETERMINATION of iron in diverse materials can be simplified and expedited by extracting the iron into an organic solvent and then determining i t flame photometrically. The iron can be extracted from a hydrochloric acid solution with 4-methyl-2pentanone and thus separated from a large number of interferences. Certain interferences that are partially extracted with the iron can be removed by back-washing the 4-methyl-2-pentanone with hydrochloric acid prior to measuring the emissivity of the iron, The 4-methyl-Z-pentanone soiution is well suited for making the flame photometric measurement; in fact, in this medium the emissivity of iron is about 20 times as intense as in an aqueous solution.
HE
1 Present address, Nuclear Materials and Equipment Corp., Apollo, Pa.
Optimum conditions were established for extraction of the iron and elimination of interferences. The spectral characteristics of iron in the 4methyl-2pentanone medium were also investigated, utilizing a modified Beckman D U flame photometer. Optimum operating conditions, the effect of interferences, and the precision of the method were established for several typical applications. EXPERIMENTAL
Apparatus. A Beckman spectrophotometer Model DU, equipped with flame sources, oxyhydrogen burner with the auxillary control panel for gas regulation mas used. For the detection and recording of the spectra, the Beckman instrument was modified by substituting t h e phototube housing and accessory power supply and recording equipment described by Kelley, Fisher, and Jones (IO).
'Pith this arrangement i t was possible to use not only the RCA 1P28 multiplier phototube with a maximum response range from 3000 to 4000 A. (S-5), but also the RCA 6217 multiplier phototube with a maximum response range of from 3000 to 6000 A. (S-10). For the determination of iron, the conimercially available recording flame photometer can be substituted. I n some of the work outlined herein the Oak Ridge Kational Laboratory recording flame spectrophotometer, &-1457A, was also used (IO). The Beckman D U atomizer-burner was modified by removing the lacquer from the adjusting screws which are used to align the palladium capillary. The adjusting screw were then silversoldered. This modification was made to ensure that the burner would function properly when organic solvents were aspirated into it. Rotameters (Brooks Rotameter Co., Landsdale, Pa.) were installed in the oxygen and fuel lines to monitor the gas
2 .o 2.5
6
15
3.0
b Figure 1. Emission of 237-mp iron line for variousoxygen and hydrogen pressures Parameters are for a given burner and may differ for each
4.0
5 10
>*
E
0
6
8
IO O X Y G E N , p.5.i.
12
16
14
VOL. 32, NO. 13, DECEMBER 1960
e
1837
flow and maintain reproducible flame conditions. Gamma scintillation counter, ORNL Model Q-1743, consisted of a DD2 linear amplifier, a built-in single-channel differential and integral pulse-height analyzer, decade scaler, and a well-type sodium iodide crystal mounted on an RCA 6655 multiplier phototube. Instrumental Setting. The instrumental settings have been described previously (11). The selection of the proper slit width is dependent upon the type of instrument, degree of reproducibility and sensitivity desired, and by the number, type, and concentration of interferences present. For the ORXL instrument, equipped with a grating spectrometer, a slit width of 0.25 mm. was used which corresponds to a spectral slit width of 1.5 nip. To achieve resolution of the 372-373.7-mp lines, a slit width of 0.15 mm. was required. The Beckman DU spectrophotometer required a slit width of 0.05 mm Fuel and Oxygen Pressures and Plow Rates. T h e optimum pressures of hydrogen and oxygen for maximum emissivity and stability of the F e 372-mp line were 2 and 10 p.s.i., respectively, as shown in Figure 1. This corresponds to a n oxygen to hydrogen flow ratio of 1 to 1. No change in emissivity was noted when the oxygcn pressure was increased to 12 p.s.i., but with pressures below 8 p.s,i. the rate of aspiration was irregular and therefore the emissivity became erratic. The variation of hydrogen pressurc had a more pronounced effect on the emissivity of the iron than changes in the oxygen pressure. The emissivity of the iron increased with increasing hydrogen pressure until up to 2 p.s.i., beyond which a sharp drop in emissivity was observed. This pronounced drop in emissivity is related t o a change in the structure of the flame which becomes larger and the base no longer is in contact with the burner. More radiation from the inner cone is, therefore, focused on the entrance slit. Whenever the fuel to oxygen ratio was changed, the posit;,on of the concave mirror in the burner housing was adjusted t o produce a maximum stable signal. Calibration Curve. Transfer I-, 2-, 3-, 4, and 5-ml. aliquots of the standard solution of iron (20 pg. per ml.) to 30-mi. separatory funnels. Add 5 ml. of hydrochloric acid and dilute each sample to 10 ml. with water. Add exactly 10.0 ml. of 4-methyl-2pentanone and slowly agitate the separatory funnels for 2 minutes. Til'ith a slow agitation rate, 30 inversions of the separatory funnel per minute, the formation of an emulsion is eliminated, without affecting the distribution ratio. After the phases have separated, transfer the organic phase to a 10- or 25-ml. volumetric flask. Measure the emission intensity of the atomic iron line and of the background at the wave lengths of 372 and 368 mp? respectively. T o obtain the net emission intensity, subtract the flame background measurement from the total ~
D
ANALYTICAL CHEMISTRY
WAVE LEhGTH rnp
Figure 2.
Flame emission spectrum of iron in 4-methyl-2-pentanone
Conditions Fe, pg./ml. Wave length region, mp Phototube, RCA Slit width, mm.
100 295 to 3 2 0 I ~2t3 0.05
340 tQ 700 6217 0.03
Relative intensities of solvent background and iron emission spectra with output from flame suppressed
PROCEDURE
Uranyl a n d Copper Sulfate Solution. Transfer a n aliquot t h a t contains from 20 to 80 pg. of iron to a 30ml. separatory funnel. Follow the procedure described for the preparation of a calibration curve. Alloys. Dissolve a 2-gram sample of each alloy (Kational Bureau of Standards No. 54b; 63a; 157) with 20 ml. of 6 N hydrochloric acid plus a few drops of nitric acid. Transfer the solutions of N B S No. 63a and 157 to 50-ml. volumetric flasks and dilute to calibrated volume with I N hydrochloric acid. Add 20 ml. of sulfuric acid t o the tin-base alloy (NBS Xo, 54b), boil, and volatilize the tin as SnBrc by the repeated addition of hydrobromic acid (9). Following the removal of the tin, transfer the sample to a 50-ml. volumetric flask and dilute to the calibrated volume with 3147 hydrochloric acid. Transfer aliquots of the sample solutions which contain from 20 to 80 pg. of iron to 30-ml. separatory funnels and extract the iron as described under Calibration Curve.
Thorium Oxide. Weigh a 2- to 4-gram portion of thorium oxide and transfer i t to a 250-ml. beaker. Add 5 ml. of 15M nitric acid, 15 ml. of 7oy0 perchloric acid, and 2 drops of 48% hydrofluoric acid and heat until the sample is dissolved. Fume t o near dryness. (Hydrochloric acid may be substituted for the nitric and perchloric acids.) After the solution cools, transfer i t to LI 100-ml, volumetric flask and dilute to 100 ml. with water. Transfer an aliquot which contains from 20 to 80 pg. of iron to a 30-mP. separatory funnel. Process it as described for the preparation of rz calibration curve, except add 1 ml. of lh' hydrofluoric acid to the hydrochloric acid backwash solution.
Flame Emission Spectra. The flame emission spectra of iron in the 4-methyl-2-pentanone medium over the wave length region of 295 t o 630 mp are presented in Figure 2. These spectra are characterized by many lines, the most prominent of which are 302.1, 344.1, 372.0, and 386.0 mp. Any of these can be selected for analytical work. At longer wave lengths, the FeQ band structures are prominent with band crests a t 561 to 565, and 622 mp. Watanabe and Kendall (IS) presented similar spectra in an aqueous medium over the region of 320 to 1100
emission intensity of the iron line. At a wave length of 372 mp the calibration curve was linear over the range tested, 3 to 50 pg. per ml. 'For convenience, 1 to 5, 2 to 10, and 10 to 50 pg. per mi. of iron were used in calibration. The coefficient of variation for these concentrations was 2%. When high concentrations of diverse elements are present, modify the procedure as follows: After agitation of the separatory funnel, drain off the aqueous phase and backwash the organic phase with 10 ml. of 5 N hydrochloric acid, which has been prequilibrated with 4methyl-%pentanone. Discard the hydrochloric acid wash solution and then aspirate the organic extract into the flame. To secure a reproducible curve, open the shutter to the multiplier phototube to the flame a t least 15 minutes before aspiration of samples to stabilize the phototube and thus minimize any drift of its output.
DISCUSSION
mp, but omitted the lower wave lengths and showed the unresolved oxide band region. Gilbert also reported on the spectra of iron in an oxycyanogen flame in the region 295 to 410 mp (8). The background due to the radiation of the organic solvent in an oxyhydrogen flame is fairly constant over the entire spectra with the exception of the band shown in Figure 2. The organic medium, therefore, does not interfere in the measurement of the most intense lines. Sensitivity and Enhancement Effect of Solvent. T h e relative sensitivity of several lines and oxide bands in aqueous and organic media is presented in Table I. Because of t h e variation of spectral slit width and t h e variance of spectral response of t h e multiplier phototubes i t is not feasible to establish an absolute scale of sensitivities. The greatest sensitivity is achieved by utilizing the spectral lines in the short wave length region. The values are in general agreement with those reported by Gilbert in his extensive table of detection limits for various eIements (7). They differ in magnitude, however, especially for the oxide bands, because Gilbert's definition of detection limits (7) differs from the base line technique used in this study. This technique, as depcribed in the section on the calibration curve, involves the measurement of relative intensities of lines and bands above the background continuum due to solvent or sample. The sensitivity of a given line or band iR expressed in terms of micrograms of iron per milliliter of final solution per scale division (0.1 mv.). When 4methyl-2-pentanone rather than an aqueous solution is aspirated, the emission of the flame spectral oxide bands and atomic lines of iron are enhanced, in most cases of the order of 10- to 2O-fold, respectively (Table I, last column). Dean and Lady observed a sixfold enhancement of the atomic spectrum of iron when acetylacetone was used as the solvent ( 5 ) . I n 4 methyl-%pentanone medium, the enhancement of the iron spectral emissimty is a t least 5 to 10 times less than that of lanthanum ( 1 1 ) . The following hypothesis is proposed to account qualitatively for this difference in enhancement of the emissivity of iron and lanthanum but it is also recognized that other factors may account for this effect. The energies of dissociation of L a 0 and FeO are 9.0 and approximately 4.8 e.v., respectively ( f ) , whereas the energies required to excite the La0-711 band and FeO561 band are 1.5 and 2.2 e.v., respectively. When a n organic solvent is substituted for water, additional energy is available which may affect the equilibrium, M O % h l and consequently the
30t /
1
E 33
20
10
-
'0
IO
Figure 3.
20 30 40 NUMBER OF INVERSIONS
50
60
Rate of extraction of iron
Conditions Solvent, 2-methyl-2-pentanone Aqueous media, 6N HCI 2 seconds One inversion,
-
relative concentration of oxide and atomic species in the flame. No dissociation of L a 0 is to be anticipated because of the relatively high energy of dissociation. The only effect is a n enhancement of the oxide band emission. I n the case of iron, however, the increase in the energy available in the flame may be sufficient to dissociate the FeO, increase the concentration of Fe and, consequently, the intensity of the
Table 1.
atomic line emission. I n fact, experimental results support this postulate. As indicated in Table I, when 4-methyi2-pentanone is substituted for water as the solvent, the ratio of the emission intensity of the FeO band, at 561 to 564 and a t 587 mp, to that of the atomic lines of the iron spectrum decreases by approximately 60%. I n the hotter organic flame the dissociation of iron was, therefore, increased. In this manner the whole spectrum of iron is enhanced but not to so great an extent as in the case of L a 0 bands. Extraction, 4-Methyl-2-pentanone is a suitable and effective medium for use in flame photometry (6, 11). Specker and Doll (19) and Claassen and Bastings (3) described the extraction of iron with 4-methyl-2-pentanone and indicated difficulties from emulsion formations. One of the authors attributed the emulsion formation to traces of grease, m;hile the other reported that it is formed in the absence of grease and recommended the addition of amyl acetate to prevent the formation of emulsions. For this reason the extraction conditions were reinvestigated by Feb9tracer, which was added to microgram quantities of iron. In this study the rate of shaking and period of time during which the shaking is continued play a dominant role in emulsion formation. If the flask is shaken slowly a t the rate of 30 inversions per minute, emulsion formation is prevented. For microgram quantities of iron the rate at which equilibrium is attained is very rapid (Figure 3), requiring only 1 minute (30 inversions). It was observed, however, that in the
Sensitivity of Iron Lines and Oxide Bands in Aqueous and Organic Media
Multiplier phototube, RCA (1P28). Volts/dynode, 60 Aqueous Organic cu.. ft./ Cu..ft./ P.s.i. min. P.5.i. min. Oxygen 10 0.16 10 0.16 Hydrogen 6 0.36 2 0.16 EnRelat,ive hnnnr. .. - - . Relative hanceSensitivity merit Normalizedb F ~ ~ hq. Org. tor6 1.6 1.6 21 1.0 1 . 0 21 1.5 1.3 24 1ImL'U'J-
I
Len Length, Wave th, $p p N
302.1 372.0 373.5 373.7 374.6 374.8 374.8
Slit Width
Background, Sensitivity, Spectral, Mv." pg./Ml./O.l Mv. Mm. -mp mp hq. Org. Aq. Org. 0.106 0.8 -1.75 2.65 4 . 0 d 0.196 0.05 0.8 -0.10 i.50 2 . 5 d 0.12' 0.05 0.8 -0.10 1.50 3.Sd 0.16' 0.05
0.8
-0.10
1.50
5.3d 0.236
2.1
0.05 0.8 -0.10 1.70 2.9d 0.14' 1.2 527.0 0.03 1.5 -0.05 0.85 89f 3.jd 36 561 5649 0.03 1.7 0 0.40 52/ 4.2d 21 5870 0.03 1.7 0 0.30 801 5.9d 32 Emission of solvent us. flame background. 6 To emissivitv of 372-mu Enhancement factor = Emissivity of E e in organic d 100 pg. Fe/ml.. Emissivity of Fe in aqueous' f 800 pg. Fe/m!. Band structure. 386.0
0
0
1.9
23
1.2
21
30
25
35
12 14
49
line of unitv. 10 pg. Fe/ml.
VOL. 32, NO. 13, DECEMBER 1960
e
1839
presence of high concentrations of other electrolytes, 5 to 10 mg. of thorium per id.--tO achieve reproducible results, the equilibration period must be extended to 2 minutes. Other systems for the extraction of Iron have been reviewed (8) and of these the ketones and phosphates esters were demcnstrated t o have the largest extraction OoeScient. The latter, because they praduce a turbuient luminescent flame, are not suitable for flame
Interference of Various Elements (Iron present, 60 w g U )
Table II.
Concn.,
Element Antimony
iron Found, %
Arg.a
i
100
105 96 98 105
20
Boron Cobalt
10 50 5
Chromium(VI)
50*
Copper
100
98
10 10 10
Molybdenum Palladiuin Rnthenium Silicon Tin Titanium Thorium
100 102 102
10
97 103 103
10 10
10 100 300
91
68 100 96 io0
3000
Uranium 90 Vanadium 3 10 Zinc 98 a In 10 mI. of solution used for measurement of radiant intensity. b NH,OHI..HCl added to reduce Cr(V1). c Organic phase backwashed with 5N HC1 0.lh' HF.
+
Table 111.
Effect of Extraneous Anions
(Iron present, 6 p g . per ml.) Concn.,
Anion Acgtate Citrate EDTA Fluoride
0.01
0.1 1.0 0.3 0.01 0.1 0.5
Perchlorate
0.01
0.1
0.5 0.5
93 88 97 98 98 98 101
98 100
90 88
65 93
99 93 79 99
84
90
13
...
APPLICATIONS
Replicate analyses for iron were made by this method of synthetic solutions simulating a uranyl sulfate: solution for use as a fuel in a homogeneous reactor and a thorium-rich solution resulting from the dissolution of a thorium oxide blanket slurry. Three National Bureau of Standards samples of alloys which differed widely as to type were also analyzed for iron by this method. S o separations other than that effected by the extraction procedure were made on a copper base alloy and a phosphor bronze. I n the analysis of a tin base alloy, the major portion of the tin was removed by volatilizing it as SnBrl from 0, sulfuric acid solution, after which the iron was extracted from a hydrochloric acid solution of the residual material and determined flame photometrically by the procedure described herein. The test results. summariaed in Ta,ble IV. had, a coefficient of variation of 4% or less.
ANALY'PICAL CHEMISTRY
Analysis of Standard Samples
.,.
... ...
.*.
66 97 96 92
..
e
Table IV.
...
1.0 75 ... 3.0 100 *. . Sulfate 0.05 97 *.. 0.75 95 ... 1.0 Tartrate 0.5 100 . Organic phase re-extracted with 10 ml. of 5N HCl prior to meaaurement of radiant intensity.
1840
flame photometric determination of iron, as Bhown in Table III. Although the remaining anions tested (acetate, citrate, nitrate, perchlorate, and phosphate) do not interfere with the extraction of iron, as evidenced from extraction tests with Fe5g tracer, these anions do interfere by quenching the emissivity of the iron. If, however, the 4-methyl-2pentanone extract is washed with 6 X hydrochloric acid prior to the measurement of emissivity, acetate, citrate, and phosphate at concentrations of 1, 0.5, and lM$ respectively, can be tolerated. However, even with the inclusion of this step, 0.1N concentration of nitrate and perchlorate cause a serious quenching of iron emission. A similar effect on the emission of iron from an aqueous medium was noted by Dean and Burger (4).
iM
1.0 0.5 0.3
HEDTA Nitrate
Phosphate
photometry. Acetylacetone, used by Dean and Lady (6) for the extraction and flame photometric determination of iron, suffers from limitations of a relatively low extraction coefficient; poorer selectivity, and greater miscibility with water. The latter condition may lead to an erratic ffame. With 4methyl-2-pentanone as the extractant the phase miscibility can be avoided if the aqueous phase is below 6 N in acid. With microgram quantities of iron the extraction is quantitative from 4 to $N hydrochloric acid solutions. T o avoid formation of a miscible phase the extractions were carried out in from 5 to 6N hydrochloric acid. Interferences. The degree to which other elements interfere with t h e flame photometric determination of iron is presented in Table 11. I n the presence of relatjively high concentration of t h e fifteen elements tested no interference is encountered with t h e exception of chroniium(V1). On reduction of sexivalent chromium with hydroxylamine hydrochloride to chromium(II1) this element can also be tolerated to a considerable extent. In the presence of a relatively high concentration of diverse substances a backwash step is required in the procedure. I n the presence of large quantities of thorium, in addition to a backwash with hydrochloric acid, i t is also necessary to use a backwash of dilute &IN hydrofluoric acid. If the last step is omitted, the residual thorium produces a luminescent flame which interferes in the determination of iron. Of the 10 anions tested as possible interferences, sulfate, fluoride, tartrate, ethylenediaminetetraacetate (EGTB or Versene) and N-hydroxylethylethylenediaminetriacetate (HEDT or Versenol 120) did not, within the concentration range tested, interfere with the
No. 54b 63a 157
NBS Standard Samples Type Tin base Phosphor bronze Copper base Synthetic Solutions
Iron, %
Present 0.028
0.52 0.053
463
U
cu
cNip 1) (3)
u
Ni
Cr(V1) Mn
10
0.5
0.030 0.52
0.054
2 2
Iron, pg. per M1.
Mg./ml.
(2)
Found
Coefficient of Variation, % 3
463
2
16.2
15.9
4
13.2
13.4
2
0.1
0.5 35 0.12
0.100 0.010
ACKNOWLEDGMENT
(3) Claassen, A., Bastinge, L., Zbid., 160, 403 (1958).
The authors acknowledge the assistance of H. P. House in the preparation of this report. LITERATURE CITED
(1) ‘‘Ayrican Institute of Physics Handbook, Dwight E. Gray, Coordinating Editor, Section 7, pp. 138-9, McGrawHill, New York, 1957. (2) Bankmann, E., Specker, H., 2. anal. Chem. 162, 18 (1958).
(4)Dean, J: A., Burger, J. C., Jr., ANAL. CHEM. 27,1052 (1955). (5) Dean, J. A., Lady, J. H., Zbid., 27, 1533 (1955). (6) Eshelman, H. C., Dean, J. A,, Menis, Q., Rains, T. C., Ibid., 31.183 (1959). (7) Gilbert,‘ P. T:,Jr.,‘Beckman Instruments, Inc., Fullerton, Calif., Bull. 753 (RiIay 1959). (8) Gilbert, P. T. Jr., Pittsburgh Conference on Anaiytical Chemistry and Applied Spectroscopy, March 6, 1958. (9) Hillebrand, W. F., Lundell, G. E. F.,
Bright, H. A, Hoffman, J. I., “Applied Inorganic Analysis,” 2nd ed., pp. 70-2, Wiley New York, 1953. (10) Kehey, M. T., Fkher, D. JTjJones, H. c.,A N A L . CHEM.31,178 (1959). (11) Menis, O., Rains, T. C., Dean, J. A,, Zbid., 31,187(1959). (12)Specker, H,, Boll, W., 2. anal. Chem. 152, 178 (1956). (13) Watanabe, H., Kendall, M. K., Jr., Appl. Spectroscopy 9 , 132 (1955). RECEIVEDfor review April 11, 1960. Accepted September 12, 1360. Division of Analytical Chemistry, 136th Meeting, ACS, Atlantic City, hT, J., September 1959.
oIorimetric Qualitative Test for Glycine R. L. SUSLETT and J.
P. JEWELL
Tennessee Polyfechnic Instifufe, Cookeville, Tenn.
b A color reaction between glycine, ethyl chloroformate, and pyridine was studied as a qualitative test for glycine. Results indicate that the reaction is specific for glycine, pyridine, and the alkyl chloroformates. This method can be adapted to spot testing for glycine with a sensitivity of 1 to 2 pgo The colored compound is stable in some polar solvents,
Hopkins ( I ) reported the formation of a red color in the reaction between pyridine and ethyl chloroformate. However, work in this laboratory has s h o m that this color depends upon the purity of the pyridine and differs from the color obtained when glycine is present. EXPERIMENTAL
T h e sources of all materials tested and their purification are indicated in Table I. The ethyl chloroformate and glycine were Distillation Products Industries (D. P. I.) White Label, and needed no further purification. The pyridine was obtained from either T h e Matheson Co. or D. P. I., White Label, and was carefully purified b y fractionation. General Procedure. Portions, 0.1 to 0.2 gram, of glycine were moistened with 0.3 t o 0.5 ml. of ethyl chloroformate and mixed thoroughly. Pyridine was added dropwise at room temperature with constant mixing. A dark green color appeared which turns red with further addition of pyridine. T h e red color can be changed to green by the addition of excess of ethyl chloroformate. Amino acids other than glycine and related compounds were tested. The results are given in Table 1. Dilute aqueous solutions of glycine were tested. A positive test was obtained if the quantity of the solution tested was less than 0.1 ml., or, if the molar ratio of ethyl chloroformate to water is greater than one. The effect of temperature and the order of addition of the reagents, glycine, pyridine, and ethyl chloroformate, are given in Table 11. I n order to determine the specificity of the reagents, alkyl chloroformates, pyridine, and compounds similar to these were substituted for the reagents in various combinations. With respect to reagents substituted for ethyl chloroformate, methyl chloroformate (D. P. I., Reagents.
react with amino acids to produce colored substances v, hich can be used for the identification and determination of these acids. The most widely used reagent in the determination of the amino acids is ninhydrin, although other organic reagents (3) and inorganic complexing reagents (2) have been used. However, none of these reagents are specific for glycine, and the identification of individual amino acids requires previous separation. o-Phthalaldehyde (4) has been found to be specific for glycine. but has never been widely used, This investigation is a study of the color reaction between glycine, pyridine, and ethyl or methyl chloroformate as a specific method of identification of glycine. When glycine is mixed with ethyl chloroformate and pyridine is added dropln-iee, a dark green color is formed which changes t o red on the further addition of pyridine or upon standing. -4 positive test is denoted by obtaining both the green and red colorations. This test combines selectivity, sensitivity, and simplicity. The sensitivity is such that it can be adapted to spot testing for glycine to 1 pg. This test also appears to be specific for pyridine and the alkyl chloroformates. Further work will determine the sensitivity and the selectivity of this reaction as a test for these reagents. AWY REAGENTS
practical) only gave a positive test. Acetyl chloride, benzoyl chloride, thionyl chloride (E. I?. I., White Label), and carbobenzoxy chloride (K &K Lab., Inc.) gave negative tests. The reagents substituted for pyridine in the test for glycine all gave a negative test. The ones used in the experiment were diethylamine, ethylenediamine, 8-quinolinol, 2- (redistilled), 3-, and 4picoline, and triethylamine (D. P. I., White Label) ; morpholine and quinoline (D. P. I., practical); diphenylamine (D. P. I., technical); pyridine (moisture present) ; and ammonium hydroxide. Diethylamine and diphenylamine produce a red coIor only. The product formed by the reaction of glycine, ethyl chloroformate, and pyridine was soluble in polar solvents. The solubility of this colored product in various solvents is given in Table 111. Pyridine and ethyl chloroformate were reacted under the same conditions as described b y Hopkins (1). Some commercial grades of pyridine gave a pale pink color, but when purified by careful distillation, no color was observed. Spot Test Procedures. A solution containing 0.1000 gram per liter of glycine r a s prepared, and then diluted to contain 10-5 gram per ml, Portions of this solutior, ranging from 0.1 to 1 ml. were pipetted onto a spot plate. The spot plate was placed in an ovcn at 135’ to 140’ C. and allowed to dry for 30 to 40 minutes. The plate was removed from the oven, and, while hot, ethyl chloroformate and pyridine were added alternately and dropwise. The colors produced were either green or red depending on whether ethyl chloroformate or pyridine was in excess. It was found that 0.1 ml. of the solut: or 10-8 gram of glycine gave a faint red or green color, while 2 x 10-6 gram of glycine gave a distinct coloration. The intensity of the coloration increased with an increase in concentration of glycine. The same results were obtained when the spot plate was allowed VOL. 32, NO. 13, DECEMBER 1960
a
1841
