Colorimetric Determination of Calcium Using Reagents of the Glyoxal Bis(2-Hydroxyanil) Class Carl W. Milligan’ and Frederick Lindstrom Department of Chemistry and Geology, Clemson University, Clemson, S.C. 29631 The determination of calcium in solution from 0.1 to 15 fig per milliliter is easy, accurate, and reproducible with reagents of the glyoxal bis(2-hydroxyanil) class. A few precautions are needed: obviously all chemicals must be calcium-free and the sequence of adding the reagents is critical. However, no extraction is needed and a simple, inexpensive colorimeter is all the instrumentation necessary. The reagents chelate calcium yielding red colors at a high pH so interferences are limited. The various reagents have been evaluated and the chelate combining ratios and the apparent formation constants measured.
GLYOXAL BIS(~-HYDROXYANIL) has been suggested often as a reagent for both the detection and the determination of calcium based on the red color produced at high pH. While this chemistry offers the promise of a simple method to learn the presence and the solution concentration of this important ion, the situation is not simple. The reagent decomposes at the necessary high pH to the amine and glyoxal; the glyoxal rearranges to glycolate anion and precipitates calcium glycolate from the alcoholic medium needed to keep the reagent in solution ( I ) . A blank error has been traced to a high level of calcium in the inorganic buffering reagents. Pure reagents were produced and found to be the solution to the blank problem. By following a strict sequence of addition: solvent, reagent, and buffer, it has been possible to obtain smooth reproducible Beer’s law plots for colorimetric methods. Also, it has become possible to apply the method of continuous variations to learn the combining ratios and chelate formation constants of seven reagent-calcium chelates. The combining ratio was one-to-one in all cases. Three new derivatives of glyoxal bis(2-hydroxyanil) have been synthesized and studied along with six derivatives synthesized earlier (2). A procedure for the determination of ultramicro quantities of calcium using GBHA was described by Williams and Wilson (3). The method was based upon the chloroform extraction of the calcium complex and was applied in the range of 0.5 to 10 kg of calcium per milliliter. The method was complicated by extraction, centrifuging, and the instability of the colored complex in chloroform. Kerr ( 4 ) developed a quantitative method for calcium in which no chloroform extraction was necessary. The water insoluble complex was dissolved in various organic solvents. Red-colored complexes were formed in aqueous-alcoholic media of pH 12.2 to 12.6. The intensities were proportional to the concentration of calcium from 0 to 40 ppm with no interferences encountered up to 50 ppm magnesium. Present address, E. I. du Pont de Nemours and Company, P. 0. Box 800, Kinston, N. C. 28501.
Umland and Meckenstock (5) postulated that the water groups in the coordination sphere mentioned by Bayer and Schenk (6) were replaced by alcohol groups. Umland and Meckenstock studied several extraction solvents and obtained the best results with a one-to-one hexanol-chloroform mixture. Using the method of continuous variations, they obtained a one-to-one calcium-GBHA complex having an apparent log formation constant of 4.3 but the pH was not given. The long-term stability of the calcium complex as reported by Kerr ( 4 ) was not observed by Vrchlabsky and Ok& (7). They theorized that the anion form of the reagent was extremely sensitive to oxidation and the reagent concentration gradually decreased. The intensity of the colored calcium complex was also observed to decrease since its concentration was always in equilibrium with the reagent concentration. Although this was an entirely plausible explanation, the present authors noted that the color faded even if the reagent was in excess. This observation led to the discovery of the mechanism of the color fading ( I ) . The present authors previously reported the synthesis of several derivatives of GBHA to study and evaluate the effect of ring substituents and their position on the performance of the derivatives as calcium reagents (2). Substitution of electron-releasing groups in the 4 position on each ring produced a reagent of much greater sensitivity than the parent compound. Substitution in the 5 position had little effect on sensitivity but greatly increased the stability of the reagent and its calcium complex. The 4-methyl derivative was considerably more difficult to synthesize than was the 5-methyl derivative or the parent compound. Substitution of electron-withdrawing groups in either the 4 or 5 position yielded stable compounds having no value as reagents since the colors formed with calcium were very transitory. Although the methylated derivatives performed better than the parent compound, the problem of the instability of the reagents and their calcium complexes still remained. For a more complete study of this problem, it was proposed that several other derivatives, in addition to those already studied, be synthesized and evaluated as calcium reagents. It was also hoped that more insight could be obtained on the chemistry of the system so that stable, reproducible Beer’s law plots could be routinely obtained. If this were done, the composition of the calcium complexes as well as their apparent formation constants could be obtained for the evaluation of these reagents. EXPERIMENTAL
Apparatus and Materials. All absorption spectra were obtained nith a Perkin-Elmer Model 4000-A recording spectrophotometer using 1-cm silica cells unless other cell lengths ( 5 ) F. Urnland and K. Meckenstock, 2. Anal. Chem., 176, 96
(1) F. Lindstrom and C. W. Milligan, ANAL.CHEM., 39, 132 (1967). (2) Ibid.,36, 1334 (1964). (3) K. Williams and J. Wilson, ANAL.CHEM., 33, 244 (1961). (4) J. Kerr, Analyst (London), 85, 867 (1960). 1822
(1960).
( 6 ) E. Bayer and G. Schenk, Cliem. Ber., 93, 1184 (1960). (7) M. VrchlabskP and A. Oki;, Collect. Czech. Cliem. Commun., 27, 246 (1962).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
are mentioned. The spectrophotometer was equipped with a P-E Model 3800 repetitive scanning attachment. pH measurements were made using a Leeds & Northrup Model 7401 pH indicator. It was standardized prior to each measurement against the buffers recommended by the National Bureau of Standards: 0.05m phthalate, pH 4.01; 0.01m borax, pH 9.18; and saturated calcium hydroxide, pH 12.42 (8). ACS reagent-grade inorganic chemicals were used in this work but unfortunately, there is no calcium specification for reagent-grade alkalies (9). Reagent-grade k e d alkalies tested contained such high levels of calcium that they could not be used. A calcium-free potassium hydroxide solution was prepared by allowing a potassium amalgam to react with water. The amalgam was prepared by the electrodeposition of a potassium oxalate solution in a Melaven cell. A spectrographic-grade carbon rod was used as the anode andredistilled mercury as the cathode. After electrodeposition, the amalgam was run off through the side arm of the cell and rinsed several times with water. The amalgam was then transferred to a well-stoppered polyethylene bottle containing water. The potassium in the amalgam slowly dissolved and the stopper was loosened from time to time to release the hydrogen gas. Potassium hydroxide prepared in the manner had a very low calcium blank. Calcium-free sodium sulfide for a buffer solution was prepared by simply recrystallizing the nine hydrate from ethanol. Ten grams of clear, colorless crystals of the sodium sulfide nine hydrate was dissolved in 50 ml of 95 ethanol at 70 "C, filtering through paper and cooling to 0 "C. The fine white crystals were filtered on a Biichner funnel, washed with 25 ml of 95% ethanol, and dried by suction until loose. The crystals were immediately transferred to a tightly-capped polyethylene bottle for storage in the refrigerator. All laboratory ware used was plastic. The calcium solutions were prepared from Mallinckrodt special primary-standardgrade calcium carbonate by dissolving in dilute hydrochloric acid, evaporation to near dryness, and dilution to volume with water. Anhydrous, acetone-free, reagent-grade methanol from Merck Chemical Co. was used to prepare the organic stock solutions of each colorimetric reagent. All water was distilled and then deionized with a column charged with Fisher Scientific Company Rexyn IRG 501, mixed-bed cation-anion exchange resin. Plastic ware was substituted for glass whenever possible to limit calcium contamination. All elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, Tenn. Glyoxal Bis(2-Hydroxy-5-Chloroanil).
z
The 4-chloro-2-aminophenol needed for this synthesis was obtained by the dithionite reduction of steam-distilled Eastman Kodak 4-chloro-2-nitrophenol. Four grams of the amine and 4 grams of Fisher 3 0 z aqueous glyoxal solution were dissolved in 100 ml of water and heated under nitrogen for 15 minutes at 60 "C. Recrystallization from ethanol yielded 2.00 grams (46.8%) of crystalline products, mp 206 "C d. A n d . Calculated for C I ~ H I O N Z O Z C ,~54.39; ~: H, 3.26; N, 9.06; C1, 22.93. Found: C, 54.58; H , 3.34; N , 9.21; C1, 22.69. (8) R. Bates, "Determination of pH", John Wiley and Sons, New York, N.Y., 1964. (9) "Reagent Chemicals," American Chemical Society, Washing-
ton, D.C., 1960.
Table I. Visible Spectral Data, Wavelength in mp Ligand, CalCiUm Complexb Reagent Xmaxa , , ,A Color 450 520 red Glyoxal bis(2-hydroxyanil) 460 520 red bis(2-hydroxy-4-methylanil) 465 540 violet bis(2-hydroxy-5-methylanil) 475 540 violet bis(2-hydroxy-4,5-dimethylanil) violet 460 540 bis( 2-hydroxy-5-chloroanil) 465 540 violet bis(2-hydroxy-5-terr-amylanil) bis(2-hydroxy-5-carbornethoxy435 505 red anil) a at pH -14. b at pH 12.5. Glyoxal Bis(2-Hydroxy-5-tert-Amylanil).
H' H ' The 2-amino-4-(terf-amyl)phenol needed for this synthesis was obtained by the nitration procedure of Close et al. (IO), followed by dithionite reduction. By nitration of Matheson, Coleman and Bell 4-(tert-amyl)phenol, the 2-nitro-4-(rertamy1)phenol was obtained in 82% yield, bp corr. 287 "C (10). The corresponding amine was obtained in 63.7% yield, mp 118-19 "C, d. Nine grams of 2-amino-4-(rert-amyl)phenol, 6 grams of 30% aqueous glyoxal solution, and 200 ml of methanol were refluxed under nitrogen for one hour. The resulting solution was poured over ice which immediately precipitated a pale yellow crystalline product. Recrystallization from methanol gave 7.00 grams (73.473, mp 193-4 "C, d. Anal. Calculated for C24H32N202: C, 75.72; H, 8.48; N, 7.36. Found: C, 75.72; H, 8.40; N, 7.38. Glyoxal Bis(2-Hydroxy-5-Carbomethoxyanil).
H,COOC
COOCH; H'
H '
By using the procedure of Cavill ( I ] ) , 3-nitro-4-hydroxymethylbenzoate was obtained; yield: 93.5 %. Recrystallization from aqueous acetic acid gave yellow needles, mp 74 "C; 74 "C (11). The corresponding amine could not be obtained by the dithionite reduction as this method not only reduced the nitro group but also saponified the ester to the unstable amino acid which quickly darkened. However, the amine was obtained quantitatively by reduction with hydrogen using platinum oxide as the catalyst, mp 90 "C; 90 "C (11). Fourteen grams of 3-amino-4-hydroxy-methylbenzoate, 8 grams of 30% aqueous glyoxal solution, and 125 ml of ethanol were heated under nitrogen for fifteen minutes at 70-75 "C. The mixture was poured over ice giving slightly brown crystals easily recrystallized from 2-methoxyethanol. Yield was 10.6 grams (71%), mp 234 "C, d. Anal. Calculated for Cl8HI6N2O6:C, 60.67; H, 4.53; N, 7.86. Found: C, 60.41; H , 4.59; N, 7.97. Chelation Reactions with Calcium. Each of the reagents synthesized in this and previous work was treated with a small amount of calcium in a methanolic solution at an appaxent pH of 12.50. The colors observed in each case are in Table I. (10) W. Close, B. Tiffany, and M. Spielman, J. Amer. Cliern. Soc., 71, 1265 (1949). (11) G. Cavill, J. SOC.Chem. Znd., 64, 212 (1945).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
1823
Table 11. Optimum pH Range Reagent Range Glyoxal bis(2-hydroxyanil) 12.20-12.70 bis(2-hydroxy-4-methylanil) 12.20-12.70 bis(2-hydroxy-5-methylanil) 12.50-12.90 bis(2-hydroxy-4,5-dimethylanil) 12.20-12.70 bis(2-hydroxy-5-chloroanil) 12,20-12.70 bis(2-hydroxy-5-tert-amylanil) 12.50-12.90 bis(2-hydroxy-5-carbomethoxyanil) 11.60-12.00 ~~
I
0
0
'
3
1
6
1
9
1
12
1
1
18
I5
1
1
21
24
1
1
27
30
1
33
'
36
1
39
'
42
45
Mlnulr8
Figure 1. Color stability of calcium-reagent chelates pg calcium (0.00025 mmole), 0.002 mmole of reagent, 0.005 mmole of sulfide as buffer, 1.5 ml of water and 3 ml of methanol n. Glyoxal bis(2-hydroxy-4-methylanil) b. Glyoxal bis(2-hydroxy-4,5-dimethylanil) c. Glyoxal bis(2-hydroxy-5-carbomethoxyanil) d. Glyoxal bis(2-hydroxyanil) e. Glyoxal bis(2-hydroxy-5-tert-amylani1) f. Glyoxal bis(2-hydroxy8-chloroanil) g . Glyoxal bis(2-hydroxy-5-methylanil)
10
1.2
I.o
.8 0
5
?
.6
? I
9
.4
.2
0
0
2
4
6
Micrograms
8
IO
12
14
16
o f Calcium
Figure 2. Beer's law plots for the recommended procedure 1 ml of Ca stock solution, 1 ml of methanol, 0.5 ml of 0.01M sodium sulfide, 2 ml of 0.001M reagent in methanol, all added in this order. Absorbance measured at the optimum time shown in Figure 1 a. Glyoxal bis(2-hydroxy4methylanil) b. Glyoxal bis(2-hydroxy-4,5-dimethylanil) c. Glyoxal bis(2-hydroxyanil) d. Glyoxal bis(2-hy droxyJ-methylanil)
Visible Absorption Spectra. The maximum absorbance wavelengths for the reagents and their calcium complexes are given in Table I as well as the color of the complexes. The spectra for the reagents were determined in a 1M potassium hydroxide solution to which a small amount of EDTA was added to mask any adventitious interfering metal ions. The spectra of the calcium complexes were determined in a methanol solution having an apparent pH of 12.50 and excess calcium. Effect of pH. The optimum p H range for each reagent can be seen inTable 11. This rangewas obtained by measuring the maximum absorbance value for each complex at the respective wavelength. Within this range, there were no 1824
Table 111. Interference of Various Ions (Micrograms of ions tolerated. 500 pg was the highest level tested) Li+ 500 Zn2500" Na+ 500 Cd2+ 500" K' 500 cu*+ 500a Mg2+ 80 Sn2+ 40 Sr *+ 10 Pb2+ 0 Ba2+ 10 SiOd2500 ~ 1 3 + 500 NO,500 uo22+
Fez+ co*+ a
0 500a 503
P043-
so,2c1-
Ni 2+ 500a c10,Cyanide added as a masking agent.
0
200 500 500
detectable changes in the maximum absorbance values for the colored complexes. Color Stability of the Calcium Complexes. To obtain the rate of formation and decomposition of each complex, 1 ml of a stock solution containing 10 pg of calcium was placed in a test tube to which had been added 1 ml of methanol. A 0.5 ml of 0.01M sodium sulfide buffer was added to give a p H as shown in Table 11. Two milliliters of a 0.001M solution of the colorimetric reagent were then added, and the absorbance spectrum was recorded periodically with the recording spectrophotometer with a repetitive scanning attachment. The rate of formation and decomposition of each complex, the sensitivity of each reagent, and the most reliable time for making absorbance measurements are shown in Figure 1 . Recommended Colorimetric Procedure-Beer's Law Plots. The Beer's law plots shown in Figure 2 were obtained in the following manner: Stock solutions containing from 2 to 14 pg of calcium were prepared. Reagent stock solutions were prepared with a 0.001M concentration of reagent in methanol. The buffer stock solution was 0.01M in sodium sulfide, 2.4 grams of the nine hydrate of sodium sulfide per liter of water, and it was stored in a polyethylene bottle. To each of seven test tubes, the following solutions were added in order: 1 ml of the sample solution, 1 mi of methanol, 0.5 ml of the 0.01M solution of sodium-sulfide buffer, and 2 ml of the colorimetric-reagent solution. Each test tube was shaken after each addition to ensure complete mixing. The absorbance of each solution was measured after the time allowed for color development shown in Figure 1. For best results, the standard solutions should be run along with a series of samples. Interference of Various Ions. To determine the interference of various ions, the recommended colorimetric procedure for calcium was applied using GBHA, the 4-methyl, the Smethyl, and the 4,5-dimethyl compounds in the presence of those ions given in Table 111. The recommended colorimetric procedure of the preceding section was used. The sample solution contained the interfering ion. To prevent the precipitatiGn of metallic sulfides when certain interferences were tested, the solution was also 1 % in potassium cyanide to mask those ions. The maximum tolerance for each ion was identical for GBHA, the 4-methyl, the 5-methyl, and the 4,5-dimethyl derivatives.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
Table IV. _-_
Component 0 1 2 3 Calciuma 0.0 0.5 1.o 1.5 Water 5.0 4.0 3.5 4.5 Methanol 0.0 1 .o 1.5 0.5 Reagent" 5.0 4.5 4.0 3.5 Equimolar calcium and reagent stock solutions were tL
Continuous Variation Mixtures (Volume in ml) Solution number 4 5 6 2.0 2.5 3.0 3.0 2.5 2.0 2.0 2.5 3.0 3.0 2.5 2.0 used to prepare each mixture.
8 4.0
I 3.5 1.5 3.5 1.5
1.o
4.0 1.0
9 4.5 0.5 4.5
10
5.0 0.0
5.0
0.5
0.0
~~
2'o I.6
f
U a I I
Table V. Apparent Formation Constants
Reagent Glyoxal bis (2-hydroxyanil) bis(2-hydroxy-4-methylanil)
Log& 4.78 5.08 bis(2-hydroxy-5-methylanil) 5.04 bis(2-hydroxy-4,5-dimethylanil) 5.26 bis(2-hydroxy-5-chloroanil) 4.40
1.2
0
n L
0 In
n 4
bis(2-hydroxy-5-tert-amylanil) bis(2-hydroxy-5-carbomethoxy-
.e
anil)
Kiz.00
4.60
pH 12 30 12.30 12.50 12.30 12.30 12.50
4.80
11.80
5.00
4.48 4.70 4.54 4.96 4.10 4.10
.4
Ca R
--
0
I.4
10
8
6 4 2 R e l a t i v e Mole P e r c e n t
0 1.2
Figure 3. Continuous variations plot for glyoxal bis(2-hydroxyanil) 1.0
Calcium plus reagent: 1.25 X lOP4M Combining Ratios and Apparent Formation Constants for Calcium and Reagent Chelates. The method of continuous variations (12) was used to determine the constants for all reagents listed in Table I. The experimental work for the calcium complexes was carried out as follows: Methanolic were prepared for each coloristock solutions of 5 x lOW4M metric reagent with the exception of the 4-methyl and the 4,5-dimethyl compounds. For these two derivatives, a 2.5 X 10-4M solution was needed. Aqueous calcium stock solutions were prepared with concentrations the same as the reagent stock solutions. GBHA, the 5-chloro, and the 5-methyl compounds dissolved readily in methanol, whereas the other derivatives would not unless they were first dissolved in re-distilled N,N-dimethylformamide before the final dilution with methanol. Various amounts of each component were added to eleven test tubes as shown in Table IV. The concentration of water and methanol remained constant in each mixture. Before the addition of the reagents, 3 to 5 drops of 1M potassium hydroxide solution were added to each test tube to give the appropriate pH values as shown in Table 11. The test tubes were quickly sealed with polyethylene stoppers and shaken to ensure complete mixing. The absorbance of each solution was measured periodically until a maximum value was obtained. The pH of each solution was then checked for a final reading. The plots obtained are Figures 3 to 9. A one-to-one complex was formed in each case. The apparent formation constant was calculated as follows. The distance from the intersection of the extrapolated straight lines to the maximum of the experimental values was divided by the distance from the intersection of the straight lines to the horizontal base line at zero absorbance to obtain the fraction dissociated designated as d. The apparent
(12) H. Diehl and F. Lindstrorn, ANAL.CHEM., 31, 414 (1959).
4
.a
0
e I
2
.6
.4
.2
CO
-
0
0
R--10
2 8
4 6
6 4
8 2
3 0
Relatlve Mole Percent
Figure 4. Continuous variations plot for glyoxal bis(2-hydroxy-4-methylanil) Calcium plus reagent: 6.25 X 10-5M
formation constant expression becomes Kh = (1 - d)/d2C, where C, is the concentration of the complex in solution assuming no dissociation and Kh is the apparent stability constant a t a given pH. The apparent stability constants are shown in Table V. RESULTS AND DISCUSSION
A high calcium blank caused much difficulty in the early phase of this investigation (2). The source of this unwanted calcium was traced t o the reagent chemicals used to adjust pH-potassium hydroxide, and sodium hydroxide. A potassium hydroxide solution was made that was calcium-free by the reaction of potassium amalgam with deionized water. The potassium amalgam was calcium-free be-
ANALYTICAL CHEMISTRY, VOL. 44, NO. l l , SEPTEMBER 1972
* 1825
1.4
1.2
1.0
a
.8
O
n L
0
n
::mi .6
ca-’
o
R - I 0
2 8
4 6 a 6 4 2 R e l a t i v e Mole Percent
16
0
R - i 0
.8
.6 0
f
“
n
a
.4
.2
-
0
Ca R - 1 0
2 8
4
6
a
6 4 2 Relativa Mole Percent
Figure 6. Continuous variations plot for glyoxal bis(2-hydroxy-4,5-dimethylanil) Calcium plus reagent: 6.25 X 10-5M
cause the potassium was obtained from the electrolysis of a clear potassium oxalate solution. The preparation of the potassium hydroxide by electrolysis was time-consuming and troublesome because only amalgams with less than 0,4% potassium could be used. More concentrated amalgams became solid. In any event, such potassium hydroxide solutions were not true buffers. A sodium sulfide solution functioned well as a buffer and solutions were completely free of calcium since Beer’s law plots passed through the origin: Figure 2. The salt was purified easily by recrystallization of the nine hydrate from ethanol. Sodium sulfide is probably calcium-free because the industrial process involves the carbon reduction of sodium sulfate at high temperature yielding sodium carbonate and sodium sulfide. Calcium is not soluble in strong solutions of either sodium sulfate or sodium carbonate. Sodium carbonate is 1826
2
4
0
Figure 7. Continuous variations plot for glyoxal bis(2-hydroxy-5-chloroanil)
Calcium plus reagent: 1.25 X lO-*M
1.0
6
R e l a t i v e Mole Percent
Figure 5. Continuous variations plot for glyoxal bis(2-hydroxy-5-methylanil)
1.2
8
Calcium plus reagent: 1.25
x
lO-4M
not soluble in ethanol as is the nine hydrate of sodium sulfide. A sodium sulfide solution also removed some interfering metal ions by precipitation as sulfides. A pleasant surprise was the absence of odor in the use of this chemical as a buffer. Above pH 12, the corrosion of even borosilicate glass becomes of concern in trace analysis and the rate of corrosion increases rapidly as the pH raises (13). It is essential that highly alkaline reagents used in trace calcium analysis be stored in plastic bottles. Calcium is a very common ion so contamination of reagents and apparatus is very troublesome. Because of the original difficulties in the use of glyoxal bis(2-hydroxyanil) as a calcium reagent, six derivatives have been prepared to see the effect various substituents and their ring positions might have on the performance of this class of reagents. According to inductive and resonant concepts, the polarity of the imino nitrogens and the phenolic oxygens should be affected by the presence of an electron-releasing or -withdrawing group on the rings. An electron-releasing group should increase the electron density in the chelation area and thereby increase the stability of the complexes with calcium. On the contrary, an electron-withdrawing group would produce a complex of lesser stability. The 4-methyl compound is more sensitive toward calcium than the parent compound. The 5-methyl compound offers greater color stability than GBHA but requires a more basic solution for the formation of its calcium complex. The possibility of increasing the number of methyl groups on the ring was tried. The 4J-dimethyl compound was an attempt to combine the sensitivity of the 4-methyl compound and the solution stability of the 5-methyl compound in the same compound, This compound reacted with calcium forming a red complex but straight-line Beer’s law plots could not be obtained and individual points were not reproducible in the earlier work (2). This led to the problem of the order of addition mentioned later. A 5,6-dimethyl derivative was unable (1 3) “Properties of Selected Commercial Glasses” Corning Glass Works, Corning, N.Y., 1963.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
to chelate calcium because the necessary planar configuration was not possible because of the methyl groups in the 6 position (2). The 4-nitro and 5-nitro compounds also reacted with calcium to give red complexes but the colors formed and vanished within ten seconds. Since these compounds were easy to synthesize, this was unfortunate, In the new series of reagents, the 5-chloro derivative was synthesized to learn the effects of a moderate electron-withdrawing group. This compound was found to chelate calcium but was of moderate value as a calcium reagent. This compound was also reported by Leminger and Farski (14) t o yield a red-violet color with calcium; they also reported the synthesis of the 4-nitro compound already mentioned (2). Since a methyl group in the 5 position produced a compound of improved color stability, a secondary or tertiary group placed in this position should yield a compound of greater stability if the groups were not so bulky that they caused steric strain in the chelate. One compound having such a structure was the 5-tert-amyl compound which reacted much like GBHA with some loss in calcium sensitivity. All reagents and calcium chelates prepared were insoluble in water so organic solvents are needed to prepare homogeneous test solutions. If the reagent had carboxyl groups, the reagent would be water soluble on the basic side of neutrality. Starting with the methyl ester of 3-nitro-4-hydroxybenzoic acid, the amine and the 5-carbomethoxy reagent were obtained in high yields. Every attempt to make the free acid from the 5-carbomethoxy compound was unsuccessful because the compound hydrolyzed at the carbon-nitrogen double bonds under saponification conditions. The 5-carbomethoxy derivative was found to chelate with calcium so it was investigated in its own right as a calcium reagent. The visible absorption spectrum of the anion form of the reagents and their calcium chelates was obtained. The spectrum of each chelate showed a single broad, intense band wellseparated from the spectrum of the reagent: Table 11. A filter colorimeter would serve well for the accurate measurement of the transmittance of the calcium chelate solutions. The band position for each calcium chelate varied slightly depending on the alcohol used. A one-to-one mixture of methanol and hexanol gave a bathochromic shift or 20 mp relative to methanol alone. Since band separation, reagent to chelate, was sufficient, no use was made of this effect. The colors produced with calcium and reagents of this class are not stable and start to fade in a few minutes, Figure 1. This fading has been noted by many workers (3,5,7) and the mechanism of the fading with GBHA and calcium has been elucidated (1). The mechanism very likely also accounts for the fading of the 4-methyl and the 5-methyl chelates. The more rapid fading of the other chelate colors may also be due to the precipitation of their calcium chelates. Alcoholic stock solutions of the reagents are not stable and darken over a period of time. The 5-methyl derivative was the most stable in stock solution. The reagents and their calcium chelates were insoluble in water so organic solvents were necessary to obtain homogeneous test solutions. When Beer’s law was applied to the 4,Sdimethyl compound, straight-line plots could not be obtained and individual plots could not be reproduced (2). It was believed that all the reagents prepared could be used as colorimetric reagents if the proper conditions could be found. In the work up to this point, the reagents were always added to an aqueous, buffered (14) 0. Leminger and M. Farskf, Collect. Czech. Chem. Commun., 30, 607 (1965).
1.2
1.0
.8
D
g
.6
0
0,
F:
n
a
.4
.2
co
-
0
R - I 0
8
6
4
2
0
Relative Mole Percent
Figure 8. Continuous variations plot for glyoxal bis(2-hydroxy-5-rerr-amylanil) Calcium plus reagent: 1.25 X 10-4M 2.0
I
I
I
I
I
I
R e l a t l v e Mole Percent
Figure 9. Continuous variations plot for glyoxal bis(2-hydroxy-5-carbomethoxyanil) Calcium plus reagent: 1.25 X 10-4M solution of calcium followed by the addition of alcohol. Using this procedure, several things were observed: The addition of the reagents to an aqueous solution precipitated the reagents and/or their calcium chelates. The addition of alcohol redissolved any precipitate that occurred, but no precipitation occurred if the alcohol was added immediately. To obtain reproducible results, an accurately timed method was needed for the addition of the alcohol because all species in solution were greatly affected by this alcohol dilution. The addition of alcohol at random times changed the reagent concentration and affected the formation constants of the calcium chelates. To eliminate this “addition error,” the colorimetric reagents were added after the addition of alcohol. This problem was solved which in turn solved many others. By strictly following the recommended order of reagent addition and using calcium-free chemicals, satisfactory and reproducible Beer’s law plots could now be obtained with ease, Figure 2, The pH for each reagent was the optimum value listed in Table 111. The time for reading each color was taken from the maximum in Figure 1. When other ions were tested in the recommended spectrophotometric method for calcium such as lithium, sodium,
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
0
1827
5.0
u
4.5
Y
er
4.0
3 .5
a-.-n
-.4
4-NOgf I
1
I
-.2
0
.2
I
I
.4 o Conatont
.6
~~
.e
I .o
Figure 10. Correlation of the log apparent formation constant for calcium chelation with the Hammett substituent sigma constants for electron density on nitrogen
a pH and in such a time interval that the reagent concentration is known not to be altered. When considering only the equilbrium formation and dissociation of the chelates and not that of the stability of the reagents, the addition of methyl groups on the ring produced chelates of higher stability than GBHA with the exception of the 5-tert-amyl compound. The log K12.0 value for this compound was expected to be large but was decreased probably due to the hindrance of the large amyl groups which may have made it difficult for the molecule to assume the necessary planar structure. The log Klz,ovalue for the 4,5-dimethyl compound was greater than either the 4or 5-methyl compound and the individual effects of the two methyl groups were combined in a single molecule yielding a stronger chelate. The addition of electronegative groups on the ring produced chelates of lesser stability than GBHA except with the 5carbomethoxy compound. The lowest log K 1 2 , ,value was observed for the 5-chloro compound and 5-tert-amyl compound. The colored chelates formed with the 4-nitro and 5nitro compounds faded so rapidly that their absorption spectra could not be recorded. It was possible to correlate the log apparent chelate formation constant with the nature of the substituent by means of the Hammett substituent sigma constants listed by Jaff6 (16). The log K12.00 values are plotted us. the u values in Figure 10. The u constants chosen are those for substituent effects on the nitrogen atom. Thus Figure 10 represents a correlation between the electron density on the nitrogen atom as influenced by the substitution. The line is a least-squares fit of the data for GBHA, the 4-methyl, the 5-methyl, the 4,5-dimethyl, and the 5-chloro derivatives. The carbomethoxy compound obviously is not behaving as part of this set. The tert-amyl compound chelate is less stable than it should be from substituent effects alone and this is attributed to steric effects. The u function for the tert-butyl group was used because reference (16) does not have a listing for the tert-amyl group. The position the nitro compounds would be expected to take is indicated, The negative slope of the line is good evidence that the nitrogens are involved in chelation. A similar approach to test substituent effects on the hydroxyl groups yielded greater point scatter and was much less useful.
potassium, aluminum, silicate, nitrate, chloride, and chlorate, they did not interfere at levels up to 500 pg, the highest level tested. The addition of cyanide also permitted levels greater than 500 pg of iron, cobalt, nickel, zinc, cadmium, and copper with no interference; these ions produced color with GBHA in the absence of cyanide. Lead and uranyl ion reacted quantitatively with GBHA producing color in this procedure for they do not form cyanide complexes. Tin interfered by clouding the solution with hydrolysis products when its level reached 40 pg. Phosphate interfered negatively by the precipitation of an insoluble calcium salt as did sulfate when its level reached 200 pg. Magnesium was tolerated up to a level of 80 pg, which is sufficient for many applications. Barium and strontium interfered when their levels reached 10 pg. The interference of higher levels of these relatively rare elements might be limited by the addition of up to 100 pg of sulfate. The results obtained are in close agreement with those of Umland and Meckenstock (5). However, the addition of sodium carbonate as recommended (5) did not successfully eliminate the interference of strontium and barium as this addition also precipitated calcium. Earlier ( 2 ) equilibrium constants could not be obtained for these reagents when the method of continuous variations was used. The method failed because the “addition error” was not known at that time. When solutions were prepared as shown in Table IV and in that order, the combining ratio and the apparent formation constant for each reagent and its calcium chelate were easily determined. A one-to-one complex was found for all reagents; Figures 3 to 9. Previously the method of Bent and French (15) was used to show a oneto-one complex for GBHA and the 4-methyl and 5-methyl derivatives. The method of continuous variations also yields the formation constants as well as the combining ratios. The apparent formation constants are given in Table V. All log Ah values were adjusted to log KI2.o by allowing a tenth of a log unit for every tenth of a pH unit difference. To explain the formation constants of the complexes, the stability of the reagents in alkaline solution must also be considered. Any change which might occur in the reagent concentration would result in a change in the complex concentration too, because these concentrations are in equilibrium. So it is necessary to carry out chelate formation studies at such
Precautions are in order to avoid contamination in the trace analysis of any common ion regardless of method. The reagents used for pH adjustment must be calcium-free. Since calcium-free reagent-grade fixed alkalies are not readily available, this can be a problem if it is necessary to neutralize a sample dissolved in acid. Samples of biological origin and ground water samples are often neutral, so many times this is not a problem. H i t is necessary to remove acid by neutralization rather than by evaporation, sodium sulfide could be used if a good fume hood was used to remove the hydrogen sulfide generated. To obtain stable, reproducible color development, it is necessary to add solutions in a definite order. This is a very simple and easy requirement to meet. The procedure would be well suited to automation where the sequence of reagent addition is locked in. The problem of interferences is limited by the high pH used which makes many cations insoluble. The sulfide anion of the buffer would also serve to limit interference and even
(15) H. Bent and C . French, J. Amer. Chem. Soc., 63, 568 (1941).
(16) H. Jaffk, Chem. Rec., 53, 191 (1953).
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
higher levels can be limited by the addition of cyanide. Samples of biological origin would not be expected to contain even those ions which cause minor interference. Different reagents have been synthesized and evaluated and the reader can choose the one which most nearly meets his needs in terms of availability, sensitivity, and color stability. In economic terms, this method does not require the expenditure of thousands of dollars for a sophisticated instrument. No time is needed for complicated alignment and adjustment. One merely mixes everything with the right reagents, the right way, and uses a simple, inexpensive, and
reliable colorimeter for calcium determinations from 0.1 to 15 pg per ml. RECEIVED for review March 7, 1972. Accepted May 12, 1972. Presented in part at the 14th Anachem Conference, Detroit, Mich., October 1966. Taken in part from the dissertation submitted by Carl W. Milligan to the Graduate School of Clemson University in partial fulfillment of the requirements for the degree of doctor of philosophy, May 1965. Work supported in part by the National Science Foundation, Research Grant No. G19699.
Quantitative Sampling from a Vertical Tube Reactor F. W. Williams and W. L. Stumpf, Jr.’ Chemical Dynamics Branch, Chemistry Division, Naval Research Laboratory, Washington, D.C. 20390 A method has been developed for quantitatively sampling the stable components of preignition events in the vertical tube reactor (VIR). Quartz microprobes using critical orifice sampling are used to achieve adiabatic cooling of the combustion processes. Since the VTR samples must often be drawn through subsequently hotter zones and the residence time in the reactor is orders of magnitude longer than in hot flame sampling techniques, additional precautions must be taken to assure sample quenching. With the proper adjustment of sampling parameters, a point source sample that is representative of the components of the precombustion reaction can be obtained for further analysis.
THE FIRST STEP in obtaining quantitative samples from a dynamic system is to withdraw a sample representative of as small a source as possible within the reactor without perturbing the chemical processes taking place. The second step is to quench any additional reactions in the sample so that subsequent analysis is representative of the chemistry taking place in the reactor and not in the sampling probe. A special problem arises when sampling a flame reactor in that samples are sometimes withdrawn through zones which are hotter than the zones being sampled. The type of reactor used in these studies was a vertical tube reactor (VTR) which is a dynamic system introduced by Williams, Johnson, and Carhart ( I ) in 1959. The unique feature of this reactor is the wide separation of the sequence of events leading to hot ignition. These include cool, blue and yellow flames and the intermediate non-luminous zones (2),each of which can be sampled separately. The particular factors related to sampling which could affect the various stages and samples are aerodynamic, concentration gradient, thermal, catalytic, and quenching. All of these effects are discussed for hot flames by Fristrom et al. (3). Several other papers have appeared in the literature Present address, Ohio State University, Columbus, Ohio. (1) K. G . Williams, J. E. Johnson, and H. W. Carhart, “Seventh Symposium (International) on Combustion,’’ Butterworths Scientific Publications, London, 1959, pp 392-8. (2) A. Fish, Angew. Chem., Itzt. Ed., 7,45 (19b8). (3) R. M. Fristrom, R. Prescott, and C. Grunfelder, Combust. Flame, 1, 102(19.57).
dealing with sampling of hot flames (4-9). In a flame, stable species are usually removed from the various flame zones by a sampling probe and analyzed with a mass spectrometer and/or gas chromatograph (IO). Another technique is to use a scavenger probe to measure unstable species such as free radicals (11). A limited number of unstable species can be measured by optical methods (12). Water-cooled probes are unable to quench reactions completely ; thus the original composition of the gas is not preserved (13). The quartz microprobe operating at critical orifice (14) has been shown to yield the most reliable quantitative data of stable species from flat flame type burners (15, 16). The sample residence time in the probe in these types of burners is relatively short (50 pec). Since the reaction zone in the VTR is 40 to 60 cm long, the sample is subjected to high temperatures for a much longer period of time. An exploratory study of sampling from the VTR in which the sample was passed through hotter zones for periods of time up to two seconds was made by Williams et al. (17). This study showed that changes in probe size and sampling speeds led to wide vari(4) J. A. Barnard and C. F. Cullis, “Eighth Symposium (International) on Combustion,” Williams and Wilkins Company, Baltimore, Md., 1962, pp 481-6. (5) R. Friedman and J. A. Cyphers, J . Chem. Phys., 23, 1875 (1955). (6) R. M. Fristrom, W. H. Avery, and C. Grunfelder, “Seventh Symposium (International) on Combustion,’’ Butterworths Scientific Publications, London, 1959, pp 304-10. (7) S. R. Smith and A. S. Gordon, J . Chem. Phys., 22, 1150 (1954). (8) S. R. Smith and A. S. Gordon, J . Phys. Chem., 60,759 (1956). (9) R. Prescott, R. L. Hudson, S. N. Foner, and W. H. Avery, J. Chem. Phys., 22, 145 (1954). (10) R. M. Fristrom and A. A. Westenberg, “Flame Structure,” McGraw-Hill, New York, N.Y., 1965, p 184. (11) Zbid.,p215. (12) W. E. Kaskan, Combust. Flame, 3,49 (1959). (13) C. Halpern and F. W. Ruegg, J . Res. Nat. Bur. Srand., 60, 29 (1958). (14) J. W. Anderson and R. Friedman, RCC.Sei. Imtrum., 20, 61 (1949). (15) W. G. Agnew and J. T. Agnew, “Tenth Symposium (International) on Combustion,”The Combustion Institute, Cambridge, England, 1965, p 123. (16) R. M. Fristrom, “Experimental Methods in Combustion Research,” Sec. 1.4, J. Surugne, Ed., Pergamon Press, New York, N.Y., 1961. (17) K. G. Williams, J. E. Johnson, and H. W. Carhart, Ind. Eng. Chem., 47,2528 (1955).
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