the tandem technique and a series of different conditions. The original chromatogram separated into four zones. The second zone contained three dyes, while the fourth consisted of two components. These mixed zones were cut out and rechromatographed under different conditions as noted. This resulted in separation of the individual dyes without eluting and reapplying t o new strips. The identity of the dyes was based on comparison of the R, values with those of the reference dyes run individually. I n the tandem procedure, modified from that of Tuckerman, Osteryoung, and Nachod (IO), the wick strip or the second-dimensional strip and dye-containing strip were overlapped 3 mm. and placed in the groove of the apparatus. An 11 X 20 mm. glass plate was placed over the overlapping zone and taped down.
Electrophoresis,” 2nd ed., Academic Press, New York, 1958. (2) Herndon, J. F., Appert, H. E., Touchstone, J. C., Davis. C. N.. ANAL. CHEM.34,1061 (1962 j. ’ (3) Hough, L., Jones, J. K., Wadman, W. H..J. Chem. SOC.1949.2511. (4) Jungbeck, S. V. F., Fachorgan Textilveredlung 15, 417 (1960). ( 5 ) Lederer, JI., Science 110, 115 (1949). (6) Maccio, I., rlnales D i m . Nacl. Qudm. 9, 52 (1956). (. 7.) Moloster. Z.. 4 n n . Chim. 3. 771 (1958). (8)Pagani, F., Tinctoria 58, 107 (1961). (9) Sivarajan, S. R., Parikh, S . G., Current Sci. (India)28, 323 (1959). (IO) Tuckerman, AI. X., Osteryoung, R. A., Nachod, F. C., $rial. Chim. Scta 19.239 (1958). (11) ’Verma, $1. R., Dass, R., J. Sci. Ind. Res. 17B,301 (1958). (12) Zahn, H., Testil-Prazis 6, 127 (1951).
Uniform transfer of solvent and solute across the joint took place. The combination of uniform solvent delivery, centrifugal force, strip groove modification in the chromatography apparatus, and the use of the tandem technique has made horizontal chromatography a useful tool in the investigation of dye separations. ACKNOWLEDGMENT
The technical assistance of A. Cowperthwaite, K. Barthalamus, IT. D . Tillett, L. Tarone, and J. C. Keyser is gratefully acknowledged. The authors are indebted to R. Davis for preparation of the figures. LITERATURE CITED
(1) Block, R. J., Durrum, E. L., Zweig, G., “Paper Chromatography and Paper
RECEIVED for revien- September 4, 1962. dccepted November 6, 1962.
Separation of Lower Aliphatic Amines by Gas Chromatography Y. L. SZE1 and M.
L. BORKE
School of Pharmacy, Duquesne University, Pittsburgh 7 9, Pa.
D. M. OTTENSTEINZ Fisher Scientific Co., Pittsburgh 79, Pa.
b A method has been developed for the gas chromatographic separation of a mixture of ammonia, methanol, and the three methylamines, using a mixture of tetrahydroxyethylethylenediamine and tetraethylenepentamine as the partition liquid. By studying the role of these two partition agents in the retention of lower aliphatic amines, the separation of a multicomponent mixture of 10 amines has been achieved. Particular attention has been paid to the elimination of the adsorption activity of the solid support.
S
of the lower aliphatic amines by gas chromatography is difficult because of support effects and the problem of obtaining the proper solvent efficiency. When dealing n i t h basic nitrogen-containing compounds there i s a tailing effect due t o the adsorption of the sample on the support. This effect is particularly acute in the case of the methyl amines. T o separate the methyl amines and ammonia, James, Martin, and Smith (5) employed a mixture of 5-ethylnonen-2-01 and liquid paraffin nith Celite 545 as the support. Burks and EPARATION
Present address, Department of Biochemistry, University of Wisconsin, Madison 6, Wis. Present address, Johns-Manville Products Corp., Manville, N. J. 240
ANALYTICAL CHEMISTRY
coworkers (1) used triethanolamine coated on C-22 firebrick, while Hughes (4) employed a mixture of n-hendecanol and n-octadecane. I n each case the deactivation of the support was not complete. James (6) further studied the separation of 27 lower aliphatic amines using two columns, one containing liquid paraffin and the other Lubrol MO (a polyethylene oxide). James, Martin, and Smith (6) washed the support, Celite 545, with methanolic sodium hydroxide to reduce the support effects. Ring and Riley ( 7 ) , Decora and Dinneen ( 2 ) , and Smith and Radford (8) reported a more effective deactivation n-ith other amine samples by depositing an alkali hydroxide on the support. I n other cases the deactivation problem was avoided by using Teflon (3) rather than the usual diatomite support. I n this study, particular attention is paid t o the deactivation of the support, the separation of the methyl amines and ammonia, and t o the general separation of the methyl-, ethyl-, and n-propylamines. EXPERIMENTAL
Equipment. Fisher-Gulf Partitioner, Model 160, modified b y placing a flash evaporator between t h e sample inlet a n d t h e column assembly. Column Materials. Chromosorb IT7, 60 to 80 mesh, tetrahydroxyethyl-
ethylenediamine ( T H E E D ) (Fisher Scientific Co., Pittsburgh, Pa.); diglycerol, technical grade and polyglycerol W (20,000 to 30,000 centipoise at 77’ F.) (Colgate-Palmolive Co., New York, N. Y.); Carbowas 400 and 1540 (Union Carbide Chemicals Co.) ; tetraethylenepentamine ( T E P ) (Eastman Organic Chemicals, Rochester, N. Y.). Reagents. All of the amines were reagent grade and were used without a n y further purification. T h e three methyl amines were received as 25% aqueous solutions and were used as such. Column Preparation. T h e liquid phase was applied to the support from a solution of a volatile solvent. T h e solvent was removed using a rotating vacuum evaporator. Potassium hydroxide was applied in a similar manner using methanol as t h e volatile solvent. T h e dried packing was placed in a 4-mm. i.d. column and conditioned for 24 hours at t h e proper temperature with a constant flow of helium through the column. RESULTS A N D DISCUSSION
Deactivation Study. Of the supports available for use, Chromosorb W was chosen because of its relative inertness as compared to Chromosorb P and because of its relatively high column efficiency as compared t o t h e Teflon type supports. Of the amines studied, the methyl amines were the
Table 1.
Per Cent Composition of THEED and TEP in Columns
TEP,
THEEZ),
Separation of methylamines A. B. C.
70
5%
1 2 3 4 5
8 6 4 2 0
0 2 4 6 8
Table II. Retention Times of Nine Amines and Ammonia” 3 4 1 2 Column 1.i 0.6 1.05 1.3 Ammonia 2.3 1.6 9.0 3.45 Methylamine 1.1 1.3 6.2 2.5 Dimethylamine 0.6 0.5 0.8 0.65 Trimethylamine 3.0 2.55 10.8 4.75 E thylamine 3.4 3.4 11.3 5.1 Diethylamine 2.4 2.95 5.1 2.9 Triethylamine 6.1 5.5 n-Propylamine 18.0 9.1 9.9 11.1 22.85 12.9 Di-n-propylamine 9.2 13.3 10.0 9.3 Tri-n-propylamine a Retention time in minutes corrected for air peak.
MLNUTES
Figure 1 .
Column
Trimethylamine Dimethylamine Methylamine
Column: 9-ft. 15% diglycerol-5% TEP on Chromosorb W: column temp.: 78’ C.; carrier gar: helium; flow rate: 100 ml./minute
5 0.65 1.8 1.6 0.65 ~.~~ 2.4 3.9 4.1 5.7 15.3 22.5
amines; however, 2y0 potassium hydroxide used with 15% Carbowas 400 or 15% Carbowax 1540 did eliminate tailing of these peaks. Five per cent THEED used as the tail reducer with 15y0 diglycerol or 15y0 polyglycerol was ineffective in eliminating tailing. Five per cent TEP proved very effective as a tail reducer when used with both 15% diglycerol and Carbowax 400. The peak symmetry obtained with 5% TEP-l5% diglycerol is illustrated in Figure 1.
1 E
I
C
most strongly adsorbed by the support as indicated by the tailing of the chromatographic peaks. Of the methyl amines, monomethyl was t h e most strongly adsorbed of the three, the dimethyl being second. Three agents, potassium hydroxide, THEED, and T E P were, in part, compared in regard to their effectiveness in deactivating the support. Two per cent potassium hydroxide used with
D
B
H
I A
I
I
J
J
A. 8.
C. D. E. Column: 15-ft. 5%
THEED-l5%
Trimethylamine Ammonia Dimethylamine Methylamine Ethylamine
F. G.
H. 1. J.
Triethylamine Diethylamine n-Propylamine Di-n-propylamine Tri-n-propylamine
TEP on Chromosorb W: column temp.:
58’ C.; other conditions same
os Figure
1
VOL. 35, NO. 2, FEBRUARY 1963
241
B E
I
5
0
10
15
20
MINUTES
Figure 3. A. B.
Separation of ammonia, methanol, and methylamines Trimethylamine Ammonia
C.
D. E.
Column: 9-ft. 15% THEED-5% same as Figure 1
Dimethylamine Methylamine
LITERATURE CITED
Methanol
TEP on Chromororb W:
Amine Separation. The separation of the methyl-, ethyl- and n-propylamines was studied using six liquid phases and combinations of these liquid phases. Initial exploratory work was carried out using diglycerol, polyglycerol, Carbowax 400, Carbowax 1540, T H E E D , a n d T E P t o separate the methyl amines and ammonia. Of the six liquid phases only T H E E D showed promise of separating the four compounds, b u t it did not give adequate deactivation of t h e support. TEP, which showed good properties as a tail reducer in the deactivation study, showed incomplete
column temp.:
60’ C.;
10- component mixture was not satisfactory. However, increasing the concentration of liquid phase to 57, THEED-15% T E P (maintaining the T H E E D / T E P ratio of 1 to 3) showed more promising separation. This is illustrated in Figure 2. For a mixture of only the methyl amines and ammonia, a somewhat better beparation than that obtained in Figure 2 can be achieved with the T H E E D / T E P ratio of 3 to 1. Using 6% THEED-2% TEP, some tailing of the ammonia peaks TVMS found, but by increasing the concentration of liquid phase t o 15% THEED-5% TEP the tailing of ammonia m s eliminated. This separation, with that of methanol, is shown in Figure 3.
other conditions
separation of the methylamines and ammonia. To study the separation of the nine amines and ammonia, five columns were prepared with varying amounts of T H E E D and T E P . These columns are listed below in Table I, and the retention time data are tabulated in Table 11. From the data in Table 11, one can choose the optimum composition of liquid phase for the separation of the 10 components. The retention time data shows that 2% THEED-6Y0 TEP provides the best separations of all 10 compounds. The actual separation obtained with this column of the
(11 Burks. R. E. .Tr.. Baker. E. B.. Clark. ‘ P., Esslinger, J.; Lace{., J. ’C. Jr.; J. Agr. Food Chem. 7, 778 (1959). (2) Decora, A. W., Dinneen, G. U., “Gas Chromatography,” H. J. Xoebels, R. F. Wall, and N. -Brenner, eds., p. 33, Academic Press, S e w Tork, 1961. (3) Fisher Scientific Co., Pittsburgh, pa., Technical Data Bulletin No. 152, Teflon as a Chromatographic Support,” May 1961. (4) Hughes, R. B., J . Sci. Food Agr. 10, 431 (1959). ( 5 ) James, A. T., Martin, A. J. P., Smith, G. H., Biochem. J . 52, 235 (1952). (6) James, A4. T., Ibid., p. 242. ( 7 ) Ring, R. D., Riley, F. W., “GasLiquid Chromatographic Analysis of Amine Mixtures,” Sixth Detroit Bnachem. Conference, Detroit, Mich., October 1958. (5) Smith, E. D., Radford, R. D., AXAL. CHEV.33, 1161 (1961). RECEIVEI)for review August 6, 1962. Accepted December 10, 1962.
Gas Chromatography of the Reduction Products
of Chlorinated Organic Pesticides HERMAN F. BECKMAN and PETER BERKENKOTTER Pesticide Residue Research laborafory, University o f California, Davis, Calif.
b A technique for the analysis of halogenated hydrocarbons with particular reference to pesticides, using a combination of gas chromatography and sodium-liquid anhydrous ammonia reduction, includes gas chromatographic isolation of the compound followed b y a reduction to liberate the halogen. Both steps measure the halogenated hydrocarbon present. A third and final step for further identification and quantitative measurement of the compound involves gas chromatographic analysis of the organic residue left after reduction. The organic phase is taken from the aqueous system into a solvent and condensed in an evap242
ANALYTICAL CHEMISTRY
orative concentrator. The residue i s then reintroduced into the gas chromatograph. The peak or peaks obtained are characteristic of the parent compound. The procedure gives three measures of a compound: its initial gas chromatography, measurement of the halogen liberated, and gas chromatography of the dehalogenated residue.
T
HE sodium-liquid anhydrous ammonia reduction technique ( I ) has been used as a means of releasing organically bound halogen for analysis. The chlorinated hydrocarbon pesticides as a group are considered t o be repre-
sentative of compounds with organically bound halogen that are generally resistant t o dehalogenation. The reduction technique would then apply to other organohalogen compounds and the subsequent analytical methods described are also generally applicable. Krzeminski and Landmann (Z), reporting on an application of the reduction procedure at the Michigan State University Conference on Pesticide Residues in the spring of 1961, described a method for the determination of chlorinated hydrocarbon residues in animal fats. The actual chloride ion detection was by potentiometric means ; however, amperometric
