The Microdetermination of Amines with 2,4-Dinitrofluorobenzene SIR: Recently v e had nccd for a sensitive micromethod for tlie quantitative deterniinatioii of some simple primary aliphatic amines. Tl'e nere not able t o obtain the accuracy and precision required in our application with s n era1 modifications of the 2, &&nitrofluorobenzene (DKFB) method (3, 7 , b i . This method consists of the reaction of tlie amine with D N F B in buffered solution to form the 2,i-diriitrophe1iylamine derivatil e (DSPA). The absoilxtiice of either tlie acidificd and diluted reaction mixture (3),or of a solvent extract of the reaction niixture (7, S), is then nieasured a t n specified navelength n ith reagent blank as reference for the spec trophotoiiietric determination. d study of these procedures revealed some source' of error, and niodific,itions :ire suggested to improve tlie :iccur:icy and precision of this method of :in:dysis. .In inherent error in the nietliod of 11cIntire ( 7 ) arises from background absorbance of 2,4-dinitroplienetole formed by reaction of DNFB n-ith ethyl alcohol, the reagent solvent specified in this method. Figure 1 shows the spectral curve, c, of 2,4-dinitrophenetole ( 2 ) (Xmas. 283 mp), and curve e of crystalline isopropylamine DNPX (Xmas. 330 mp) each with cyclohemne as solvent and as reference. Curves a, b, and d represent increasing concentrations of isopropylamine in the reaction mixture and shorn that the amount of phenetole formed is variable. Figurc 2 illustrates the effect of variable phenetole formation on the precision of the analysis conducted awording to N c Intire. The spectral curves are of cyclohexane extracts of replicate reactions identical in quantity of amine and reagmts used. The variable absorbances a t 283 nip show that the amount of phenetole formed is variable and gives rise t o uncertain contributions to the DNPX absorbance a t 330 mp. This uncertainty occurs in both the reagent blank and amine samplcs, but n-ould not b~ of importance with secondary amines d i o s e DKPA derivatives hare Xmax. in the region of 350 t o 360 mp. In this region the contribution to absorbance by 2,4-dinitrophenetole is negligible. Rosenthal and Tabor (8) reacted the amine nith DKFB in a more dilute system, extracted the acidified reaction mixture nith polar solvent (cyclohesanone, ethyl acetate, or methyl iso-
butyl ketone), and measured the absorbance of the extract. Dubin (5) eliniinated the extraction step and measured tlie absorbance of the reaction mixture diluted with HC1-dioxane. I n both procedures the lack of desired precision may be attributed to the presence of excess DNFB, dinitrophenol, or both in the solutions !Those abeorbances are being measured. Thus, DNPA absorbances are read from high background levels and steep slopes. In Figure 3, spectral curve a is a reagent blank while curves b and c are tn-o levels of isopropylamine in diluted reaction mixtiires according to the procedure of 1.0
,
I
1
11
4:
260
280
300
340 380
460
Wavelength in millimicrons Figures 1-3.
Spectral curves
Beckman DK-2 ratio recording spectrophotometer with 1 -cm. cells
Dubin ( 3 ); hydrochloric acid-dioxane is reference for each curve. Analyses by the method of Rosenthal and Tabor (8)resulted in similar types of curves when pure solvent was the reference. To overcome the difficulties observed above, n e recommend that no D N F B reactive solvents such as alcohols be used in the analysis; excess DSFB be completely hydrolyzed by strong a1b:ili or, in situations where high pH values are undesirable, reacted n ith glycine (5, 6 ) ; and that the dinitrophenylaiiiine be extracted froni the alkaline aqueous reaction mixture into a n immiscible solvent such as cyclohexane for the spectrophotometric determination. \Ye have used purified dry dioxane (9) as solvent for DNFB to avoid formation of the phenetole. This re:igent is stahle for several weeks a t room temper:itwe. TVhen this reagent is used and the malj-sis completed by the procedure of NcIntire (Y), the absorbances of reagent blanks (spectro grade cyclohexane reference) are zero in the region 2i0 to 460 mp. Furthermore, spectral curves of cyclohexane extracts of the reaction mixtures are identical to thobe of the pure crystalline derivatives ( I ) dissolved in cyclohexane with ma\ima near 330 nip for primary amines (Figure 1, e) and near 355 mp for secondary amines. As all background from excess reagent or by-products is eliminated, replicate analyses give identical results, and rigor in the preparation of the reagents is not required. Cyclohexane extracts of amine-DNFB reactions prepared according t o recommendations outlined m-ere compared spectrally with solutions of the corresponding pure crystalline D S P A . Recoveries of n-butylamine, isobutylamine, and 2-aminobutane were quantitative. Recoveries for isopropylamine and tert-butylamine were 85% and SO%, respectively, even when the reaction was carried out in sealed tubes with excess DNFB reagent. However, straight line absorbance-concentration plots were obtained using standard procedures. I n our application of the analysis to biological systems, we often found it necessary to remove ammonia prior to reaction with DNFB. The published method for ammonia remoiTal with mercuric oxide without loss of amine (4, IO) was satisfactory. During trials to check ammonia removal with VOL. 34, NO. 4, APRIL 1962
0
583
mercuric oxide, the partition coefficient of 2,Cdinitroaniline (DNA) between the water-diluted reaction mixture (7) and the extracting cyclohexane was about 8 in favor of the alkaline aqueous phase. This partition suggested a means of removing ammonia after the DNFB reaction, with the elimination of the mercuric oxide separation. The saturation concentration for DNA in the cyclohexane phase under the conditions of analysis is about 13 pg. per ml. and would be the maximum that could be found in the cyclohexane phase. In a 1-em. cell with cyclohexane reference, this DNA concentration corresponds to a n absorbance of 1.03 (hmas., 315 mp), The absorbance a t 330 mp (hmax. DNPA primary amine) of a cyclohexane solution of DNA was reduced from 0.60 to 0.045 with a single extraction using an equal volume of 0.5M aqueous sodium bicarbonate and to 0.005
with a second extraction. One-tenth normal HCl, 0.1N NaOH,0.2M NaZC08, and 0.1M NaHZP04gave similar results with no solution being superior to the others for this purpose. The derivatives of 2-aminobutane, isopropylamine, and tert-butylamine, the only ones tested, did not extract into the aqueous phase. An alkaline extractant is preferred to avoid the possibility of partitioning 2,4dinitrophenol into the cyclohexane. This separation procedure is simple and rapid for nominal quantities of ammonia. Large quantities of ammonia would consume excessive amounts of the DNFB reagent. LITERATURE CITED
(1) Asatoor, A. M., J . Chromatog. 4, 144 (1960). (2) Buttle, B. H., Hewitt, J. T., J . Chem. SOC.95,1755 (1909).
(3) Dubin, D. T., J . Bid. Chem. 235, 783 (1960). (4) Francois, M., Compt. rend. 144, 567 (1907). C.A. 1, 1697 (1907). (5) Grassmann, R., Hormann, Endres, H., Ber. deut. chem. Ges. 1477 (1953). (6) Lockhart, I. M., h’ature 177, 393 (1956). (7) McIntire, F. C., Clements, L. M., Sproull, Muriel, ANAL.CHEM. 25, 1 757 (1953). (8) Rosenthal, S. M., Tabor, C. IT., J. Pharmacol. Exptl. Therap. 116, 131 (1956). (9) Vogel, A. I., “A Textbook of Practical Organic Chemistry,” 2nd ed., p. 175, Longmans, Green, Kew York (1951). (10) Weber, F. C., Wilson, J. B., J. Biol. Chem. 35, 385 (1918).
M. J. KOLBEZEN J. W. ECKERT BRIGITTE F. BRETRCHNEIDER
Department of Plant Pathology Citrus Research Center University of California Riverside, Calif.
Further Improvements in the Preparation and Utilization of Tetra butyla mmonium Hyd roxid e Titra nts SIR: Since the publication, in 1956, of our paper (1) on the preparation and utilization of tetrabutylammonium hydroxide as a basic nonaqueous titrant, numerous investigators have employed this or similar titrants to analyze acidic materials and to differentiate acid mixtures. Riddick (4, 6) summarized a large portion of this work in his biennial reviews. Cundiff and Markunas ( 2 ) observed that there were impurities in the titrant which led to erroneous results in resolutions of acid mixtures containing strong acids. The impurities, although unidentified, were removed successfully by passage of the titrant through a short anion exchange column. This anion exchanged titrant appeared satisfactory when used in 0.1N strength, but anonialies were noted with the more dilute titrants, particularly on extended storage. The anion exchange step also adds considerably to the preparation time. Therefore, it seemed desirable to investigate more closely the preparative means to determine if sources of impurities could be eliminated. Possible impurities in the tetrabutylammonium hydroxide titrant include tributylamine, tetrabutylammonium carbonate, and dissolved silver oxide. In a recent study, Marple and Fritz (3) investigated the source of amine and carbonate impurities and reported techniques for their removal. Our studies ( 2 ) have indicated that the amine impurity does not contribute substantially 584
e
ANALYTICAL CHEMISTRY
sufficiently pure tetrabutylammonium iodide is used in the synthesis. The most likely impurity is tetrabutylammonium carbonate, with dissolved silver oxide as suspect minor impurity. Much of the carbonate impurity was introduced through use of commercially available silver oxide in the synthesis. KO such material tested was completely free of silver carbonate. Since tetrabutylammonium hydroxide is an excellent carbon dioxide absorbnnt, the remainder of the carbonate impurity came from handling and transfer steps in the preparation. The recommended method for preparation of 0.1N tetrabutylammonium hydroxide is as follows:
shaking. Add 900 ml. of benzene to the flask, reflush with nitrogen, restopper, shake vigorously, and replace in the salt-ice bath for 15 minutes. Filter under a nitrogen atmosphere through a h e porosity sintered-glass funnel. Transfer the filtrate to a 1-liter volumetric flask, allow to warm to room temperature, and dilute to volume with benzene. Allow to stand for 24 hours, and if any silver oxide separates, refilter. Store the titrant in a reservoir protected from carbon dioxide and moisture. Prepare 0.02N tetrabutylammonium hydroxide by dilution of 200 ml. of the 0.1.Y titrant and 30 ml. of methanol to 1 liter with benzene. Prepare other dilute titrants using proportionate amounts of 0.1N titrant, methanol, and benzene as for the 0.02N titrant.
Dissolve 30 grams of reagent grade AgNOs in 50 ml. of carbonate-free water, add 55 ml. of 4N carbonate-free NaOH, shake vigorously, then filter under a nitrogen atmosphere through a medium porosity sintered-glass funnel. Wash the precipitated silver oxide with 760 ml. of boiling water, then with 300 ml. of methanol. Dissolve 40 grams of tetrabutylammonium iodide (Rymark Laboratories, Terre Haute, Ind.) in 95 ml. of methanol in a 1-liter flask. Chill the solution in a salt-ice bath (-5’ to - 10’ C,), then add the freshly precipitated, methanol-moist silver oxide. Flush the flask with nitrogen, tightly stopper, and replace in the salt-ice bath. Allow the reaction to proceed for 1 hour with frequent, vigorous shaking, replacing the flask in the salt-ice bath after each
This means of preparation precludes formation of tetrabutylammonium carbonate, and the solubility of silver oxide in the chilled methanol-benzene is negligible. Tests for sodium, nitrate, halide, and carbonate were all negative on all preparations evaluated. The use of high purity tetrabutylarnrllorlium iodide and the low reaction temperature negate the possibility of amine forniation. Titrants prepared in this manner are stable and maintain constant normality for at least 30 days a t room temperature, if protected from carbon dioxide. The reference electrode previously recommended (1) for use in these non-
to anomalous results, particularly if
