Table IL Carbon Balance in Fluorination of Pentaerythritol"
Found Compound Cc. CF, 29.7 CZFG 1.5 n-CaF8 0.9
Converted to CF,, Carbon, Cc. 29.7 3.0 2.7
52 5 5
T o h l carbon in sample as CF1
=
.57.42 cc.
'
lowering the effective trap temperature have not been successful. The amount of carbon tetrafluoride found seems to be affected more by the method and rate of opening the reactor to the vacuum system than by the equilibrium vapor concentration. However, a correction for the carbon tetrafluoride content can be made rather easily as previously described. An attempt was made in one of the runs in which all of the oxygen mas recovered from pentaerythritol to obtain a carbon balance. The temperature of the traps wvas increased to -73" C.
and several fractions 15 ere collected. The fractions were analyzed by infrared absorption using a Perkin-Elmer double-beam spcctrometer and by making comparisons with spectra of the pure compounds available in the literature. Table I1 shows that 62% of the carbon was recovered in the fractions collected. The rest of the carbon undoubtedly occurs as less volatile compounds. Because of possible damage to the glass measuring system by the corrosive fluorinating materials, i t was not feasible to increase the trap temperatures to obtain a more complete carbon recovery. Even under the severe conditions of fluorination, the carbon skeleton had not been completely disrupted. This method for direct determination of oxygen in organic compounds is easily carried out in relatively simple apparatus. Since volatile carbonyl fluoride is a frequent intermediate in the fluorination of organic compounds, it is essential t h a t the reaction vessel be shaken vigorously to assure completion of the reaction. Carbonyl fluoride is not readily fluorinated in the gas phase and thorough mixing with the molten
fluorinating reagent is necessary. Because the sample oxygen is not diluted with other oxygen, the method should have particular utility in oxygcn exchange studies for oxygen not readily exchangeable with carbon dioxide. LITERATURE CITED
Ayscough, P. B.,Can. J . Chem. 33, 1566 (1055). Banks, A. A , , Emrleus, H. ,J., Koolf. -4. A,, J . Che/?i.,Soc.1949, 2861. Elving, P. J., Ligett, IT, R., Chem. Rem. 34, 129 (19-14). Grosse, .4.V., Hindin, S.C , Kirshenbaum. A . D.. A X A L . CIIEM. 21, 386 (1949).
'
Hoekstra, H. R., I h t z , J. J., Zbid., 2 5 , 1608 f19.531.
Lee; - T . ' S . i - ~ e y e r ,R , - ~ n a l .Chim. Acta 1 3 , 3 4 0 (1959).
lleyer, F. R., Rouge, G., 2. angezo. C h e m 52, 637 (1939).
Sheft. I.. LIartin. A . F . I h t z . J . J.. J. Am: Chem. SOC. 78.' 1557 (1956): ( 9 ) TVoolf, A. A,, Emeleus, H. J., J . C h e m SOC.1949, 2865.
RECEIVED for review Xovemher 16, 1956. Accepted April 20, 1957. Division of .4nalytical Chemistry, 130th Xeeting, ACS, Atlantic City, N. J , September 1956. Based on work performed under the auspi,ces of the E. S. Atomic Energy Commission.
Quantitative Determination of Histamine in Presence of Certain Interfering Metallic Ions A. C. ANDREWS and T. D. LYONS Department of Chemistry, Kansas State College, Manhattan, Kan.
b Various metallic ions interfere with colorimetric methods used to determine histamine concentrations. A method is presented to nullify this interference utilizing diethylenetriaminepentaacetic acid. A comparison of the stability constants for histamine-metal ion complexes with corresponding diethylenetriaminepentaacetic acid-metal ion complexes indicates that the metal ions are almost completely held in the latter complex. This complex allows the histamine to b e determined without detectable error.
A
the chemical methods utilized for the quantitative determination of histamine, @-imidazoyl-4-ethylamine, colorimetric procedures appear to be the most sensitive. These methods depend chiefly upon the light absorbance of the coupling product of histamine and a diazonium ion in alkaline media or the reaction product of histaVOKG
mine R ith dinitrofluorobenzene (5, 7 ) . Substituted phenyldiazonium ions t h a t have been used successfully include p-sulfonic acid ( 2 , S ) , p-bromo- ( I j 63, and p-nitro- ( I O , 11). Certain metallic ions, principally cobalt(II), copper(I), copper(II), nickel(II), and iron(III), cause interference in each of the above methods by forming a stable complex with histamine (8, 9). This metallic complex resists the coupling reaction with a resulting decrease in the concentration of histamine which niay undergo these reactions. A procedure has been outlined ( 7 ) to nullify the interference of trace amounts (microgram ion) of these metals in the analysis involving dinitrofluorobenzene which utilizes 8-quinolinol or sodium diethyldithiocarbamate to eliminate the interference. The present paper introduces a new method to nullify the interference as i t occurs in the coupling procedure involving the p-sulfonic acid diazonium ion.
Preliminary studies were carried out on various chelating agents including 8-quinolinol, sodium diethyldithiocarbamate, sodium cyanide, and diethylenetriaminepentaacetic acid (DTPA). 8Quinolinol was unsatisfactory because it undern-ent a reaction with the diazonium ion 'i\ hich r e d t e d in a high blank reading. Sodium diethgldithiocarbamate was unsatisfactory because it formed a cloudy solution with high absorbance n i t h the diazonium ion. The cyanide ion proved moderately satisfactory for lo^ concentrations of histamine, Diethylenetriaminepentaacetic acid proved entirely satisfactory, showing no interaction with the diazonium ion and completely nullifying the metal ion interference over a wide range of histamine concentrations. EXPERIMENTAL
Reagent grade materials were used throughout and solutions were prepared with distilled water. Histamine diVOL. 29, NO. 9, SEPTEMBER 1957
1325
phosphate, G5H1~N3P208, was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. and diethylenetriaminepentaaretic acid was supplied by the Geigy Chemical Corp., New York, under the trade name Chel 330. The histamine was dried and weighed in a wcuum tube which was constructed t o suspend from one arm of an analytical balance. Metal ion solutions were prepared from cupric chloride, nickel nitrate, and cobaltous nitrate; the metal ion concentrations were determined by electrolytic deposition (4). The Beckman Model I>U spectrophotometer was used with 1-em. matched cells maintained a t 20" C. The wave length of maximum absorbance for the coupling product of p sulfonic acid phenyldiazonium ion and histamine had been reported (2) as occurring at 500 mp, this was verified in the present work. The p-sulfonic acid phenyldiazonium chloride reagent was prepared as follows: A stock solution of sulfanilic acid hydro-
t
Table 1. Determination of Histamine in Presence of M e t a l Ions a n d Diethylenetriaminepentaacetic Acid Metal Ion
Copper(I1) 25.0 25.0 50.0 50.0 50.0 50.0 100.0 100.0 Cobalt(11) 29.0 29.0 .58 0
58 0
58 0 *58 0
116.0 116 0
Histamine Concn., Moles/ Liter x 106 Taken Found Difference 2.50 8.21 1.82 5.34 6.90 7.94 4.94 14.83
2 8 2 5
62 12 02 47 6 94 7 87 4 87
+o
20 +0 13 +0 04 -0 07 -0 07
1 1 61
-0 22
2 52 8 32
+o 02 $0 11 +0 10 +o 09 -0 08
2.50 8.21 1.82 5 34 6.90 7.94 4 94
$0 12 -0 09
1 .5 (5 8
92
43 82 05
4 98
14.83
14 67
2.50 8.21 1 82 5 31
2 69
+o
11 +0 04 -0 16
Sickel( 11)
22.0 22.0 14 0 44 0 44 0
44 0 88 0
88 0
Figure 1 . Histamine determination in p r e s e n c e of certain metallic ions .1. DTPA in presence or absence of certain metallic ions B. 2.9 x lO-4M Co(I1) without DTPA C. 2.5 x 10-4.1.1r Cu(I1) without DTPA
6 '30 7 94 4 94
14 83
8 3.5
2 24
3 77 7 12 8 17
4 86
14 i 5
+0 19 +o 14 +o 42 4 0 43 +o 22 +O 23 -0 08 -0 08
chloride was prepared by dissolving 0.047 mole of sulfanilic acid monohydrate in 9 ml. of concentrated hydrochloric acid and diluting to 100 ml. This stock solution gave consistent results for 6 months. Into a 50-ml. volumetric flask immersed in ice was introduced 2 ml. of the stock solution followed by 6 ml. of 0.72M sodium nitrite. After this mixture had stood for 5 minutes, 6 ml. of sodium nitrite was added and the solution \vas diluted t o 50 ml. The reagent, which mas kept in an ice bath, was ready to use after 15 minutes and was stable for 12 hours. The coupling reaction was carried out in 10-ml. mixing cylinders. Five milliliters of 1.2% sodium carbonate, 0.021M in diethylenetriaminepentaacetic acid, was introduced, followed by 2 ml. of histamine solution whose concentration range was 5 X 10-6M to 2 X 10-4M. (This volume may be decreased accordingly for more concentrated solutions. It is essential that all metal ions be in the form of the diethylenetriamine~
~~
~
~
T a b l e II. Precision of Histamine Determination Using Diethylenetriaminepentaacetic Acid Solution NO.
Histamine Concn. Found, Moles/Litrr x 10;
Av. Dev. of
Detn., P.P.M
pentitacetic acid complex hefore the diazonium solution is added.) The contents were mixed, 2 ml. of the diazonium solution was added, and the contents were agitated quickly and traneferred to the spectrophotometer. Readmgs were taken every 15 seconds a t 500 mp with the slit width set at 0.03 until a maximum absorbance was observed. This usually required about 2 minutes bnt varied slightly with the histamine cnncentration. In Figure 1 the interference caused by metal ions is shown. I n B and C the diethylenetriaminepentaacetic acid has been omitted from the determination, while A shows the results of the determinations carried out with diethylenetriaminepentaacetic acid as outlined. The average molar absorptivity is 6.74 X lo3 liters mole-' cm.-l for histamine solutions using diethylenetriaminepentaacetic acid in either the presence or absence of metal ions (curve -4). Table I shows the accuracy with which histamine concentrations may be determined in the presence of metal ions. Table I1 presents the precision which may be obtained on quadruplicate determinations using the same procedure as used in Table I but in the absence of metal ions. DISCUSSION
6
1326
14.18
14.09
ANALYTICAL CHEMISTRY
14.11
14.01
0.48
The use of diethylenetriaminepentaacetic acid permits the determination of
I-iist:trnine to be carried out in the presence of relatively large amounts of certain metal ions. The stability constants (log units) for the metal complexes of diethylenetriaminepentaacetic acid (12) n i t h copper(II), nickel(II), and cobalt(I1) are 21.03, 20.21, and 19.00, respectively, M hile the corresponding stability constants for histamine (9) are 16.03, 11.91, and 8.95. This R oultl indicate thst a very small concentration of histamine as a metal complex exists in equilibrium with diethylenetrianiinepentaacetic acid This allons for a greater yield of the coupling product hetween histamine and p-sulfonic acid phenyldiazonium chloride than would be obtained if the histamine F l a y largely in the form of a metal complex. The maximum concentrations of metal ions n1iic.h may be tolerated for
copper(I1) and cobalt(I1) are 3.0 X 10-zM and 7.2 X 10-3AM‘respectively. ACKNOWLEDGMENT
The authors wish to thank the Public Health Service, National Institutes of Health, for Grant E-l354(c2) for a portion of the funds necessary t o carry out this work, and Dowell, Inc., Tulsa, Okla., for a fellowship to T. D. Lyons. LITERATURE CITED
(1) Barand, J., Genevois, L., RIandillon,
G., Ringenbach, G., Conzpt. fend.
222,760 (1946).
( 2 ) Hnvinya, E., Seekles, L., Strengers, Th., Jr., Rec. trav. chim. 66, 605 (19.27). (3) Iloessler, I
