Chromatographic Separation of C1 to C b Primary and C2 to C12Secondary Monoamines as 3,5-Dinitrobenzamides DELWIN P. JOHNSON and JAMES B. JOHNSON Development Department, Union Carbide Chemicals Co., Division o f Union Carbide Corp., South Charleston, W. Va.
b Primary and secondaty monoamines
REAGENTS AND APPARATUS
are separated as their 3,5-dinitrobenzamides by elution analysis on silicic acid columns using n-hexaneethyl ether mixtures as developing solvents. Identification is made from Rf values obtained after locating the zones b y treating eluate fractions with acetone and aqueous potassium hydroxide to form quinoid ion colors. R, values are listed for all normal primary and secondary species of methyl through hexylamines.
Adsorbent. Two parts of Mallinrkrodt's Chromatographic-~Iad~silicic acid were thoroughly mixed with one part of Hyflo-Supercel and stored in a sealed container. Solvents. %-Hexane (Phillips Petroleum Co.) was redistilled and the hearts fraction used for this work. Ethyl ether (Union Carbide Chemicals Co.) W'RS uscd without additional purification.
A
investigation of the analytical aspects of liquid phase chromatography revealed relatively little work on amines. This may be due t o the comparative ease with which the lower amines can be analyzed by gas chromatography or gas-liquid partition chromatography, or t o the limited ability of tertiary amines to form satisfactory reaction products. Fuks and Rappoport (2) reported the use of partition chromatography on starch to separate ammonia and mono-, di-, and trimethylamines. Clayton and Strong (1) separated members of the homologous series of primary monoamines on Celite filter with a solvent system consisting of methanol-water as the immobile phase and petroleum ether as the mobile phase. Ry incorporating phenolphthalein in the stationary solvent, the individual zones were followed as pink bands on the white column. Van Duin (6) prepared the colored 2,4-dinitrobenzene derivatives of primary and secondary amines and separated them on silica gel; the zones appeared as yellow bands. A project was initiated in this laboratory to investigate the use of liquid phase chromatography for resolving amine mixtures, Jvith the hope of extending the principle to compounds with boiling points exceeding the limitations of gas chromatography. It was discovered that the 3,5-dinitrobenzamides of primary and secondary amines possess properties which permit both separation and identification of the parent amines. GENERAL
Solvent A. One volume of hexane plus 2 volumes of ether. Solvent B. T M Ovolume\ of hexane plus 1volume of ether. Solvent C. Five volumes of hexane plus 2 volumes ot ether. Solvent D. Three vohimes of hexane plus 1volume of ether. Acetone, reagent grade or redistilled commercial grade. 3,5 - DinitrnhcnzoJ-lchloride, reagent grade. Trimethylaniiiic, commercial grade (Union Carbide Chemicals Co.). PREPARATION OF 3,5-DINITROBENZAMIDES
The method of Shriner, Fuson, and Curtin (4) for the preparation of substituted benzamides was modified for preparing the 3,Sdinitrobenzamides. Triethylamine was substituted for pyridine as proton acceptor to facilitate crystallization of the lower amine products. The higher amines did not produce easily crystallized products. Consequently, the latter n'ere analyzed on the residue obtained by evaporating the benzene extract of the reaction mixture. ANALYSIS
A 25-ml. buret with a cotton plug was employed as the chromatographic tube and the column was packed dry with the aid of slightly reduced pressure on the tip of the buret. The adsorbent retention volume, V,, was determined by adding a measured quantity of solvent to the dry column and subtracting the amount remaining after the adsorbent was saturated. Ether, being more strongly adsorbed, proceeds through the dry column somewhat more slowly than hexane and can be followed as a warm band due to
heat of adsorption. The column qhould not be charged with the sample until sufficient fresh solvent has passed through to ensure temperature equilibrium. Approximately 1.5 mg. of each of the, amides was dissolved in 1 ml. of solvent and 0.3 ml. of the solution \vas introduced into the column with pressur?. After walls of the buret had been rinscd n-ith 0.3 ml. of solvent, the chromatogram mis developed with the aid of nitrogen pressure a t a flow rate of 1 drop per second and the eluate \vas collected in 0.3-ml. fractions. The calibration of the buret served conveniently for measuring the fractions. The zones were located bv adding 2 ml. of acetone and 2 drops of 10% aqueou. potassium hydroxide t o each fraction. A characteristic quinoid ion color developcd in fractions containing the amides. R , values were calculated by using the formula V,/V, = RI. The elution or threshold volume, V,, for each amide was determined by multiplying the number of the first fraction in each zone by 0.3 nil. DISCUSSION
The niechanim of the reaction of m-dinitro and trinitro aromatic compounds with acetone and a strong alkali to form colored quinoid ions has been discussed in some detail by Porter (3). The effectiveness of this reaction appears to be influenced by the nature of substituenta, other than the nitro groups, on the benzene ring. For example, esters of dinitrobenzoic acid and the dinitrobenzaniides form fairly stable colors whereas the colors from the amine salts of the acid are unstable. The 3,5-dinitrohcnzamides of primary amines produce less stable colors than the secondary amine products. Whereas the colors from secondary derivatives may remain without apparent change for as long as 30 minutes, those from the primary derivatives gradually become purple and within a few minutes appear almost red. This effect serves as an excellent means of characterizing the parent amine; in some instances when zones overlap on the chromatogram, this color change can be used as VOL. 31, NO. 8, AUGUST 1959
1373
Table I.
Amine Methyl Ethvl Dimethyl Diethyl n-Propvl n-But,d n-Am$l n-Hexyl Di-n-propyl Diallyl Di-n-butyl Di-n-amyl Di-n-hexyl
R,
of adsorbent or solvent to ailother. Standard Rl values shouId, therefore, be established on each new lot of material used. Gradient Elution. Considerable “tailing” was exhibited by the substances having low R, values, and, in some instances, caused the zones to extend over a n excessive number of fractions. To prevent this condition, the principle of gradient elution was applied. Although tailing was significantly reduced and separations were good, considerable skill is required to reproduce R, values satisfactorily with gradient elution.
Values of 3,5Dinitrobenzamides
Solvent -4
R I Values Solvent B Solvent C
0.40 0.63 0 27 0 63
0.14 0.22 . 0.09 0.30 0.37 0.47 0.55 0.61 0.63 0.63 0.79 0.88 0.93
Solvent D
~~
0 25
0.43
0.28
0.59
0.40 0.55 0.76
0.84
Rfizi!I
PRIMARY
Table
II.
Resolution of Mixture Containing Five Amines
Color after
15 Minutes
Blue Purple
RJ 0.79 0.63 0.47 0.37 0.22
Identity Dibutyl Dipropyl Butyl Propyl Ethyl
020
CARBON ATOMS
Diethyl Butyl Dipropyl Dibutyl
Retention Volume, 8, R , 8.8 8.6 8.8 11.3 6.1 8.8 8.4 8.8 8.4 8.8 11.3 6.1
0.22 0.22 0.29 0.30 0.30 0.47 0.46 0.63 0.62 0.78 0.79 0.79
a fairly accurate indication of the front of the succeeding zone. Established R f values for the amines tested are listed in Table I. Contrary to expectations, the R, value for the dimethyl derivative is lower than that for the monomethyl derivative. The curves in Figure 1 were constructed by plotting the R, values in Table I against the number of carbon atoms in the parent amines. For each solvent two curves are shown: one for primary and the other for secondary amine derivatives. By observing the color stability to determine the species and using these curves to determine the number of carbon atoms, a fairly positive identification of an unknown amine can be made. The 3,5-dinitrobenzamides were prepared from a mixture containing ethyl-, propyl-, dipropyl-, butyl-, and dibutylamines, but the products failed to crystallize. Instead, an ambercolored, highly viscous material was obtained upon complete evaporation of 1374
ANALYTICAL CHEMISTRY
0.8
04
Table 111. Reproducibility of R, Values on Columns of Various Lengths
Derivative Ethyl
SECONDARY
2
4
6
8
0 1 2
CARBON ATOMS
Figure 1. Relationship of R, values to number of carbon atoms in alkyl radicals of N-substituted 3,5-dinitrobenzamides 0
A
Solvent A Solvent B
the benzene. This material \\as chromatographed using solvent B; five distinct zones appeared. As illustrated in Table 11,the fire zones were identified with those anlines present. To demonstrate the reproducibility of the R, values. the 3,5-dinitrobenzamides were chromatographed with solvent B on columns varying in length from 10 to 20 em. The results are shown in Table 111, where V , indicates the various column lengths. Slight variations in R, values were observed in changing from one batch
LITERATURE CITED
(1) Clayton, R. A,, Strong, F. M., h - . 4 1 > . CHEM.26, 579 (1954). (2) Fuks, x. A . , RapPoport, M. A , Doklady Akad. Nauk, U.S.S.R. 60, 1219 (1948). (3) Porter, C. C., AXAL.CHEX 27, 805 (1955).
(4) Shriner, R.L., Fuson, R. c., Curtin, D. Y., “Systematic Identification of Organic Compounds,” p. 226, Wiley, New Yo&, 1956. (5) Van Duin, H., Biochirn. Biophys. A4cta 12,490 (1953).
R~~~~~~~for review November 13, 1958, -4ccepted April 23,1959.
Precision Absorptiometry-Correction In the article by Crayton AI. Crawford on “Precision Absorptiometry” [AKAL.CHEM.31, 343-8 (1959)], column 2, page 344, a paragraph of text was omitted from the section on “Application to Concentration Ileasurement” and printed in error as a correction a t the end of the article (page 348). It should have appeared as a paragraph of that section, and is repeated here as follows: Reilley and Crawford [ANAL.CHEM. 27, 716 (1955)l showed that power
losses which are a constsnt fraction of Po-for example, as caused by reflection a t the cuvette front surface-lead to the same results, if they are consistently either taken into or left out of account. The symbol P conform‘ to
the recommendation of the Joint Conimittee on Komenclature in Applied Spectroscopy [ANAL. CHEX 24, 1349 (1952)]. although I is nearly universally used. A symbol for Definition 4 has not been standardized; Davies and Manning [ J . Am. Chem. SOC.79, 514s (1957)l used p , which was the original choice of Reilley and Cralvford but \vas changed by typing difficulties. In the same article, three typographical errors have been noted. On page 346, middle column, line 22: Equation 15 should be Equation 5; line 30: dt should read dT; line 3 7 . (29) should be (28).
=111 of the above corrections have been niade in the author’s reprints.
