(11) Grassmann, W., Hormann, H., Endres, H., Chem. Bcr. 86, 1477 (1953). (12) Hirs, C. H. W., Ann. N . Y . Acad.
- -
scz. - - -. S8.R11(1SflO). - , - - - .- .,
(13) Hirs, C. k. W., Moore, S., Stein, W. H., J . Biol. Chem. 219,623 (1956). (14) Inn, H.R.,Manske,. F.,. J . Chem. Soc. . i926,-2348. ' (15) Levy, A. L.,Nature 174, 126 (1954). (le) Lewis, J. C. Snell, N. S., Hirschmann, D. J., draenkel-Conrat, H., J . Bid. Chsm. 186, 13 (1950). (17) Nystrom, R. F., Brown, W. G., J . Am. Chem. SOC.70,3738(1948).
(18) Porter, R. R., Melhods in M e d . Research 3 , 256 ( 1950). (19) Ramachandran, L. K.,Biochim. et Biophys. Acta 32, 557 (1959). (20) Ramachandran, L. K., McConnell W.B., Nnlure 176,931 (1955). (21) Rothm, A., J . Gen. Phyaiol. 24, 203 ( 1940). (22) Sanger, F., Biochem. J . 39, 507 (1945). 123) Smith. E.L.. Kimmel. J. R.. Brown. * D, M., Thompaon, E. 6. P., ' J . B i d : Chem. 215,67 (1955).
(24) Smith, E. L.,Stockell, A , , Kimmel, J. R.. Zbid.., 207., 551 (1954). (25) Thompson, E. 0, P.; Ibid., 207, 563 (1954). (26) Wagner-Jauregg, T.,Short, J. H., Chem. Ber. 89 253 (1956). (27) Zahn, H., bfanrnllller, IT., Biochem. 2.330,97(L958). RECEIVED for review Novemhcr 29, 1DflO. Acce ted March 21, 1961. 1nveRt.igation aided) by a grant from the Rockefeller Foundation.
Spectrophotometric Determination of Iron with N,N-Bis(carboxymethy1)anthranilic Acid DONALD L. DINSEL and THOMAS R. SWEET Deparfment of Chemistry, McPherson Chemical laboratory, The Ohio Sfafe University, Columbus
b Previous investigation of the derivatives of anthranilic acid in this laboratory has shown that N,N-bis(carboxymethy1)anthranilic acid forms a colored complex with iron. A new method for the spectrophotometric determination of iron has been developed which involves the addition of ferric iron to an excess of N,N-bis(carboxymefhyI)anthranilic acid between a pH range of 1.4 to 1.6. The absorbance is measured at 370 mp.
D
Young (2) observed that cobalt(I1) formed a pink-colored complex with N,N-bis(carboxymethy1)anthranilic acid at p H 7. It was reported that the complex has a n absorbance maximum at 525 mp, and that the complexes of zinc, cadmium, mercury, and nickel did not absorb at this wave length, but that the yellow complex of ferric iron had a n absorbance curve which increased steeply in this region. Attempts to mask the yellow color with fluoride ion were unsuccessful because a precipitate believed to be salts of the complex FeFa appeared. Experiments were performed to develop a method for cobalt using N,Nbis(carboxymethy1)anthranilic acid, but the attempts were not successful because results were not reproducible and the cobalt complex was unstable. However, this reagent could be used for the spectrophotometric determination of iron(II1) at a wave length of 370 mp. At this wave length there is a large absorbance difference between the iron complex and the reagent at a point where the absorbance of the reagent is small (Figure 1). The absorbance of the reagent becomes very large as the wave length is decreased below 370 m l . Absorbance measurements were URING INVESTIGATIONS,
1078
ANALYTICAL CHEMISTRY
IO, Ohio
tion by addition of solid potassium hydroxide. The reaction misture was stirred constantly. After 5 hours a n additional 80 to 100 grams of chloroacetic acid were added to the reaction 08 beaker. Sufficient potassium hydroside was also added to keep the solution basic to litmus. The solution was then stirred for an additional 3 hours and allowed to remain overnight in the reaction vessel. The following morning the solution was cooled in an ice bath and made acid to a p H of about 2 with 1 to 1 hydrochloric acid. After further chilling, the orange precipitate was removed by suction filtration and dissolved in 1 to 1 boiling acetic acid. The hot solution was filtered and allowed t o cool in an ice bath. The supernatant liquid was removcd by filtration and the precipitate was dissolved in hot distilled water. At this point activated charcoal was added to decolorize the impure WAVE ILENGTH, MILLIMICRONS reagent. After filtration to remnve the charcoal, the solution was cooled in an Figure 1. Absorption curves ice bath, and a white material precipitated from the solution. Again this preI . iron, reagent, and sufficient nitric acid to adlust the p H to 1.5 vs. a reference blank containripitate was dissolvcd in hot distilled ing reagent and nitric acid. Solution was 64.4 water, filtered, cooled, and refiltered. p.p.m. with respect to iron. Concentration of This procedure was repeated. reagent was 0.01 2M The material at this stage of purifica2. Reference blank far curve 1 vs. water tion had a melting range of 208' to 3. Nitric acid vs. water 209' C. This is below the desired melting range of 210' to 212' C. The most probable impurity is the N-caralso made at 390 mp for comparison boxymethylanthranilir acid which is less since the reagent does not absorb at soluble in water than the reagent. this wave length. The impure material was dissolved in boiling water, removed from the hot EXPERIMENTAL plate, and allowed to cool without Reagents. N,N-BIS(CARBOXY- chilling. Some of the material crystallizrd. This precipitate was removed METHYL)ANTHRANILIC ACID. T h e reafrom the warm solution and discarded, gent was first prepared by Volander and the filtrate was cooled in a n ice and Mume (1) and then by Young (2). bath for about an hour. The remaining The procedure used here is essentially material crystallized and was filtered that used by Young with several modiand washed sevcral times with small fications to obtain larger quantities. portions of cold distilled water. The Anthranilic acid, 137 grams, and material was dried in a vacuum desiccachloroacetic acid, 230 grams, were added tor over phosphorus pentoxide. The to 1800 ml. of distilled water. The dried product obtained by the above mixture was made basic to litmus and procedure had a melting range of 210' maintained basic throughout the reac-
"'h
1 0 -60 r
I
g,,,
mp
050-
p
Table
Interfering Ion Ni
390 m!J
040-
-a
m
co
0
8
a
030-
cu
020-
I
i
8,
0.10 0
I
I
I
2
3
Cr
4
PH
Mg K
Figure 2. Variation in absorbance of iron complex with PH
Mn
Solutions were 64.4 p . p m wlth respect to Iron
Zn Pb c1
to 212' C. with decomposition. CAL CULATED: C, 52.10; H,4.37; N, 5.53; equivalent weight, 84.5; FOUND: C, 52.01, 51.94; H, 4.60, 4.55; N, 6.52, 5.60; equivalent weight, 84.4.
N,N
Results of Interference Studies
Weight, Mg. 0.59 2.91 4.69 0.39 1.96 3.14 9.82
Fe Found,
Concn., P.P.M. 23.6
Mg. 1.62 1.65
116 ~~-
i.68
187 15.6 78.4 126 393 6.2 25 126 64.8 129 648 640 1280 800 4000
o.i3
0.64 3.17 1.62 3 24 16.216.00 32.00 20.00 100.00 4.00 16.00 16.1 16.1 1.41 3.50 36.5 20.00
1.61 1.61 1.61 1.64 i.62 1.70 2.16 1.61 1.62 1.63 1.61 1.61 1.61 1.61
1m
_..
1.61 _-
640 644 644 56.4 140 1460 800
1.62 1.61 1.61 1.61 1.61 1.68 1.61
- BIS(CARBOXYMETHYL)ANTHRApH
ACIDSOLUTION.A 0.012M solution was prepared by wei hing 3.038 grams of the acid and a d i n g it to a liter flask. About 750 ml. of distilled water were added, and the flask waa placed in a 60' C. water bath for 20 minutes. It waa then shaken in an elcctric shaker until the remaining acid went into solution. After cooling to room temperature the solution waa diluted to the mark with distilled water. STANDARD IRON SOLUTION. Pure iron wire (99.8% Fe) was polished with a piece of fine emery cloth, wiped, and cleaned in acetone. The wire was placed in a 100-ml. beaker. A slight excess of concentrated nitric acid was added slowly, and the solution was heated to remove the oxides of nitrogen. Periodic additions of distilled water were made to maintain the liquid volumc. Thr iron solution was transferred quantitatively to a 500-ml. volumetric flask and diluted to the mark with distilled water. Several standard iron solutions were prepared in this manner. Their concentrations varied from 500 to 803 p.p.m. Apparatus. All p H measurements were made with a Beckmnn Model G NILIC
so4
I.
meter e uipped with microelectrodes. A I quantitative absorbance measurements were made with a Beckman quartz spectrophotometer, Model DU, equipped withha Beckman photomultiplier attachment, Model 4300. For measurements between 320 and 400 mp, a red-pur le Corex filter was used. Corex ce Is, 1 cm., were used, ahd a slit width of 0.06 mm. was maintained during all measurements. Other absorbance measurements were'made with a Cary recording spectrophotometer, Model 14. Working Curve. Standard iron solutions were measured according t o the following procedure: Various amounts of standard iron solution (805.3 p.p.m.; p H = 0..5) were pipetted into each of seven 25-ml. volumetric flasks, and 10 ml. of 0.012M solution of reagent were added t o each flask. When necessary the p H was adjusted with nitric acid to the range between 1.4 and 1.6. The solutions were diluted to the mark with reagent. The absorbance was measured against a reference solution which
P
Error,
Abs. Error, Mg. 0.01 0.04 0.07 0.00 0.00 0.00 -0.07 0.01 0.09 0.55 0.00 0.01 0.02 0.00 0.00 0.00 0.00
9%
0.60 2.48 4.31 0.00 0.00 0.00 -4.31 0.60 5.59 34.2 0.00 0.60 1.24 0.00 0.00 0.00 0.00 0.00 0.60 0.00 0.00 0.00 0.00 4.31 0.00
0.00
0.oi
0.00 0.00 0.00
0.00 0.07 0.00
contained, in place of the iron, the same volume of nitric acid a t p H 0.5, the same amount of nitric acid used to adjust the pH, and the same amount of reagent. This procedure was adopted because the nitrate ion absorbs slightly at 370 mp aa shown in Figure 1. All concentrations of iron rccorded are the concentrations of iron in the final solution-Le., milligrams of iron per 25 ml. of solution. It is advantageous, when preparing an iron solution, to adjust the p H of the solution to approximately 0.5. When aliquots of this solution are added to the reagent, the resulting solution will have a pH which is very close to the optimum range for maximum absorbance. Thus, little adjustment is necessary to obtain the ideal conditions of PH. DISCUSSION
As shown in Figure 2, the absorbance of the complex is dependent on pH. The absorbance remains constant over a range of 1.4 to 1.6. All absorbance
e
n
-
-
0800/ 0800
-
O 0 V 040 II
0 0
I
I
1
2
I 4
I 3
I 5
I 6
J7
PH
080 I
120 I 1.20
I60 II
200 lI
240 l1
280 II
320 1 1
3M I ,
400
MOCAR RATIO REAGENT TO IRON
Figure 3. Variation in absorbance of reagent with pH
Figure 4. Variation in absorbance of iron complex with molar ratio of reagent to iron at constant PH
Solutions were 0.01 2M wlth resped to reogent
Solutions were 128 p.p.rn. wlth respect to Iron
VOL. 33, NO. 8, JULY 1961
1079
0 600
I
I
I
I
I
I
I
I
,370mll
0803 --
0 700
.-
0600
-
0--0~:37Ohp-
D - - a 4=390rnp
0
3
"
0
" 4
~
'
8
I
I
I
I
I
I2
16
20
24
28
.32d
TIME, HOURS
Figure 6. with time
Variation in absorbance of iron complex
Solution was 64.4 p.p.rn. with respect to iron
0
020
040 060 080 MOLE FRACTION
100
Figure 5. Results obtained with Job's method of continuous variation
readings were taken within this range. Since the reagent also absorbs slightly at 370 mp the effect of pH on its absorbance was studied. Figure 3 shows that the absorbance a t 370 mp decreases with a n increase in pH. However, in the range in which absorbance measurements were made, 1.4 to 1.6, the absorbance is constant. Figure 4 ehows the effect on absorbance of a n increase in the amount of reagent. The curve starts to level at about a molar ratio of 1.2 and becomes constant as the ratio approaches 4. Thus absorbance measurements were always made with the reagent in excess of a t least four times the amount of iron present. Job's method of continuous variation showed that the iron and reagent forms a complex in the ratio of 1 to 1 (Figure 5 ) . As shown by Figure 6, the absorbance remains constant for
about an hour and then a slight increase is registered in the absorbance. The increase over the first hour is evidently too small to detect. Readings were taken within 30 minutes of preparation to avoid any error due to the time factor. Interferences were studied by the addition of known concentrations of various ions to iron solutions of 64.42 p.p.m, (1.61 mg. per 25 ml. of iron). The iron was then analyzed according to the above procedure. The interfering ions were added until the amounts were at least 10 times the amount of iron present or until an appreciable error--i.e., 4%--was recorded. Table I shows the resultg of the interference study. Zinc, lead, chromium, and magnesium cause no significant interference when present in amounts 10 times the iron concentration of 64.42 p.p.m. Copper interferes a t 5 p.p.m., while cobalt begins to interfere between 126 and 392 p.p.m. and nickel a t about 60 p.p.m. Potassium and magnesium offer no interference at concentrations of 4000 and 1280 P.PJ% respectively. sulfate ion gives no interference and chloride interferes only a t a concentration of about 1400 p.p.m., probably because of the formation- of yellow feiric chloride. Except for cobalt, a]] of the ions which interfered caused an increase in the absorbance. Copper is the most serious
of the interfering ions and cannot be detected visually in the iron solution. Hence, if present in concentrations above 5 p.p.m., it should be removed before proceeding with the analysis. The proposed method providea an accurate and rapid method for the spectrophotometric determination of iron. Total iron is determined, since any iron originally present aa ferrous iron would be oxidized by the nitric acid present. The color formed is stable up to an hour. There is no need for a stabilizing agent. The complex of iron which is formed is soluble in water. The reagent is not available commercially but can be made rather easily. The method is not aa sensitive as many spectrophotometric methods for iron, but it does have a wide range of a t least 8 to 96 p.p.m. The sensitivity is 0.68 p.p.m. of Fe. This is taken as the concentration of iron needed to produce a change in absorbance of 0.005. LITERATURE CITED
(1) Volander, D., Mume, E., Der. deul. chem. Ges. 33, 3182-3 (1900). (2) Young, W., Ph.D. thesis, Ohio State Unlvemityj lg5&
for review December 21, 1960. Accepted April 17, 1961. Taken in part from the M.S, thesis of Donald L, Dins& Ohio State University, 1960. RECEIVED
Determination of Acid Sites on Solid Catalysts by Ammonia Gas Adsorption R. T. EARTH and E. V. BALLOU' Gulf Research and Development Co., Pitfsburgh, Pa.
b A new apparatus and procedure are described for the determination of the amount of ammonia gas adsorption on catalytic materials at elevated temperatures. The results obtained can be related directly to the amount and relative strength of surface acid sites on solid catalysts. Applications of the method to various catalysts are pre1080
ANALYTICAL CHEMISTRY
sented, along with a general survey of the technique and system. The data are shown to b e reproducible and to give adsorption curves characterizing various catalyst materials.
T
measurement of surface acidity on catalysts has been of considerable interest to the petroleum processing HE
industry. A relationship h a s been noted between surface acidity and cracking or polymerization activity (3,4). Also, basic nitrogen compounds have a poisoning effect on catalysts in processing units. These considerations have 'Present address., Missiles and Space Division, Lockheed Aircraft, Sunnyvale, Calif.
