The detail in which the results are reported in Table Iv indicates the excellent separations and extreme semitivity that are possible. This detail is valuable for following the change in individual hydrocarbon concentrations
(2) California Motor Vehicle Pollution Control Board, "California Test Pro-
as the dilute exhaust gas is irradiated in in the smog chamber.
R. w., in “Gas Chromatography,” N. Brenner, J. E. Callen, M. D. Weise, eds., p. 423, Academic Press, New York, 1962. (5) Greene, S. A., Pust, H., ANAL.CHEM. 29, 1055 (1957). (6) Heaton, w. B., Wentworth, J. T., Zbid., 31, 349 (1959). (7) Hurn, R. W., et al., Proc. Am. Petrol. Inst. IZZ 42, 657 (1962). (8) Innes, W. B., Bambrick, W. E., Andreatch, A. J.. ANAL. CKEM.35, 1198 (1963). (9) Jones, J. L., et nl., J . Air Pollution Control Assoc. 13, 73 (1963). (IO) Martin, R. L., ANAL. CHEM.34, 896 (1962). (11) Mayrsohn, H., O’Neal, Q., Division of Water and Waste Chemistry, 148th Meeting, ACS, Chicago, Ill., September 1964.
ACKNOWLEDGMENT
I am grateful to T. J. Prater, who operated the gas chromatograph, and to J * D* Benson, M* and W. hl. Wiese, who provided exhaust gas samples and helpful suggestions. D. E. Seizinger, Bureau of Mines, provided the mercuric perchlorate packing. LITERATURE CITED
(1) Brownson, D. A., Stebar, R. F., SOC.
Auto. Eng. Meeting, Chicago, Ill., May 17-21, 1965.
cedure and Criteria for Motor Vehicle Exhaust Emission Control,” Revised Jan. 23, 1964. (3) Coulson, D. M., ANAL. CHEM.31, (lg5’)‘ (4) Ferrin, C. R., Chase, J. O., Hurn,
(12) McEwen, D. J., ANAL,CHEM.35, 1636 (1963). (13) Zbid., 36,279 (1964). (14) McEwen, D. J., J . Chromatog. 9, 266 (1962). (15) Nebel, G. J., Jackson, M. W., J . Air Pollution Control Assoc. 8 , 213 (1958). (16) Neligan, R. E., Mader, P. P., Chambers, L. A., Zbid., 11, 178 (1961). (17) Perkin-Elmer Corp., Norwalk, Fonn., May “Model 1962. 800 Instrument Manual, p. 5, (18) Rowan, R. Jr., ANAL. CHEM. 33, 658 (1961). (19) Scott, C. G., in “Gas Chromatography, 1960,” R. P. W. Scott, ed., p. 317, Butterworths, Washington, 1960. (20) Scott, C. G., J. Inst. Pet. 45, 118 (1959). (21) Smith, R., Rose, A. H., Jr., Kruse, R., Int. J. Air Water Poll. 8 , 427 (1964). (22) Stephens, E. R., Pattison, J. N., Division of Water and Waste Chemistry, 144th Meeting, ACS, Los Angeles, Calif., April 1963.
RECEIVED for review February 21, 1966. Accepted April 25, 1966.
Determination of Micro Quantities of the C1-C4 Primary and Secondary Amines by Electron Aff in ity Detection EDGAR W. DAY, Jr., TOMASZ GOLAB, and JOHN R. KOONS
Eli M y and Co., Greenfield laboratories, Greenfield, Ind.
b A method utilizing thin-layer and gas chromatography for the separation, identification, and determination of the 1 1 C1-C4 primary and secondary amines is described. The amines were reacted with 2,4-dinitrofluorobenzene and the excess reagent was destroyed. The resulting dinitrophenylamines were partially separated into three distinct zones by thin-layer chromatography. The zones were removed from the plate, the adsorbent was eluted with organic solvent, and the eluate was analyzed by gas chromatography using an electron affinity detector. The column packing was 2% diethyleneglycol succinate on a silanized solid support. Complete separation of all of the amines was obtained and most of these can b e determined a t a concentration of 1 .O pg./mI. or less in aqueous solutions.
T
HE DIKITROFLUOROBEKZENE (DSFB)
method of RIcIntire, Clements, and Sproull (17) is a simple and sensitive method for the determination of primary and secondary amines in aqueous solutions. In this method, the amines are converted to the corresponding N substituted dinitroanilines (DNPamines) which are measured spectro-
photometrically a t about 330-350 mp. The reagent blank is relatively high by McIntire’s procedure but the modifications of Kolbezen, Eckert, and Bretschneider (12) reduce this blank. The method offers no selectivity without separating the amines either before or after dinitrophenylation. Separation of the volatile free amines is impossible by paper or thin-layer chromatographic techniques, but some separations using the hydrochlorides can be obtained (4, 7 , 20). Chromatography of the DXP-amines seems to offer some advantages and has been studied by others (2, 7 , 16). The DNP-amines are nonvolatile, soluble in organic solvents, and readily removed from the adsorption medium for further measurement. It is doubtful that the chromatographic separation of all of the Cl-C4 amines can be accomplished unless several different systems are employed. Gas chromatography has been employed for the separation of aliphatic amines in several instances. For the free amines, basic liquid phases or alkaline-coated supports are required to obtain separations ( 1 , 13, 18, 21). The more volatile amines are difficult to handle, however, and various techniques have been utilized to liberate the free base from the hydrochloride
salts a t some point in the gas stream (‘7,11). To our knowledge, no attempts have been made to separate the DNPamines by gas chromatography. Vanden Heuvel, Gardiner, and Horning (22) studied the gas chromatographic characteristics of 14 different derivatives of the C12-C14 amines but not the DNP derivatives. And Wilk et al. (84) used the heptafluorobutyramide derivatives to determine some biological amines by gas chromatography. Solutions containing 1.0 pgJm1. of amine can easily be analyzed by the spectrophotometric DNFB method. Gas chromatographic methods using thermal conductivity or flame ionization detectors can not approach this sensitivity. Aromatic nitro compounds, however, are highly sensitive to detection by the electron affinity detector. Landowne and Lipsky (16), for example, have analyzed amino acids by methylation and dinitrophenylation prior to gas chromatographic assay with an electron affinity detector and achieved a high degree of sensitivity. Thus, preparation of a suitable column should facilitate a highly selective and sensitive method for amines. The development of such a method is described in this communication. The amines are reacted with DNFB, the VOL. 38, NO. 8, JULY 1966
1053
Table 1.
NO.
Melting Points of DNP-Amines
Amine Methylamine Ethylamine n-Propylamine Isopropylamine n-Bu tylamine sec-Butylamine Isobut lamine &Butyfamine Dimethylamine Methylethylamine Diethylamine
1 2 3 4 5 6 7 8 9 10 11
Melting point ( O C.) Observed Literature 178 -180 114 -115.5 96.5- 9 7 . 0 94.0- 9 4 . 5 89 - 91 54.5- 5 5 . 0 78.5- 8 0 . 0 154.5-155.0 84.5- 86 53 - 57 67 68
Table II. Gas Chromatographic Instrument Parameters for DNP-Amines
Temperatures Column Injector Detector Nitrogen flow
190° c. 250' C. 210' c.
Recorder Applied Potential output
1.0 mv. 26 volts D.C. 1 . 0 X 10-0 A.F.S.
Approximately 100 ml./min.
(amperes full scale)
excess reagent is destroyed, and the DNP-amines are extracted and determined by gas chromatography with an electron affinity detector. All 11 of the C1-C4 derivatives cannot be resolved by gas chromatography alone. Partial separation by a thin-layer technique is required to accomplish complete separation and determination.
-
1054
ANALYTICAL CHEMISTRY
(1,10)
80 153-153.5 86- 87 55 79- 80 69
(2, 6 )
(2, 23)
(2, 6 ) (2, 6, 23) (9, 6, 23)
(6) (23) (9)
( 6 , 23) (2, 3 )
Gas Chromatography. All gas chromatograms were obtained on a JarrelAsh Model 28-710 universal gas chromatograph equipped with an electron affinity detector. The column consisted of 0.25-inch o.d., 5-mm. i.d., borosilicate glass tubing, 6 feet in length, packed with 2% diethyleneglycol succinate (DEGS, Applied Science Laboratories, Inc., State College, Pa.) on Gas Chrom Q (Applied Science), SO/lOO mesh. An all-glass injection system was employed. The other instrument parameters are listed in Table 11. The temperature of the column and the flow of nitrogen are somewhat variable depending on the quality of the packing, but the values listed were generally suitable. All of the compounds were dissolved in benzene and diluted to the desired concentration in the same solvent. All
Figure 1. Thin-layer chromatogram of DNP-amine mixtures A. E.
Compounds 2, 3, and 7 (See Table I) Compounds 6 and 9 C. Compounds 4 and 10 D. Compounds 1 and 5 E. Compounds 1-1 1 Inclusive
of the chromatograms shown were obtained from solutions containing 3.0 ,ug./ml. of each derivative. The volume of solution injected was 1.2-1.5 fil. from a 5-pl. Hamilton syringe. For quantitation, direct standards were used for references and the responses were determined by measuring the peak heights in centimeters. The sensitivities of the compounds were determined by the procedure of Landowne and Lipsky (14). The noise
10-
9-
EXPERIMENTAL
Preparation of Standard Materials. The standard DNP-amines were prepared from commercially available amines or amine salts. About 1.0 gram of amine was dissolved in 50 ml. of borate buffer solution (see below), D N F B (2 ml. in 25 ml. of p-dioxane) was added and the mixture was heated on a steam bath for 1 hour. After adding 50 ml. of 2 N NaOH, the mixture was heated for another hour to hydrolyze the excess reagent. After cooling, the solid was filtered, washed with 0.1N NazC03 and recrystallized from ethanol-water or petroleum ether-cyclohexane. The lower melting derivatives did not solidify in the reaction mixture. These mixtures were extracted with hexane, which was washed with O.1N NazC03, dried with anhydrous Na2S04,and evaporated to dryness on a rotating vacuum evaporator. The residue was then recrystallized from petroleum ether-cyclohexane. The melting points of the materials prepared are listed in Table I with some literature values. There seems to be some uncertainty in the correct value for N,Ndiethyl-2,4dinitroaniline and no value could be found for the secbutylamine derivative.
Reference
179-180 113-114 95 94- 95 89- 91
a76LLI
m Z
0 5-
a
EU 4-
3-
21-
0-
MINUTES
Figure 2. amines
Gas chromatogram of the 1 1 Cl-Cd primary and secondary DNPNumerals refer to compounds listed in Table I
10-
98-
7-
W
6-
2
g
5-
W
E
4I
7
3
4
5
6
7 8 9 IOII..
3-
Figure 4. Thin-layer chromatogram of the individual DNP-amine standards
2-
See Table I for compound identification
1-
CTT-T-
0-
1
MINUTES
Figure 3.
Gas chromatogram of the DNP-butylamines (Zone Ill Standards) See Table I for peak identification
level of the system employed was about 4 X lo-'* amp. The minimum sensitivity was then defined as the amount of component required to give a peak height of three times the noise level or 1.2 X 10-l1 amp. Experimentally, the recorder was set to give full scale deflection a t a 1.0 x 10-10 amp. and solutions of the compounds were injected to give a peak height of about 1201, of full scale. This amount, in moles, divided by the peak width a t the base line in seconds, yielded the sensitivity values in moles per second. Thin Layer Chromatography. Standard 20- x 20-em. glass plates coated with a 250-micron layer of Silica Gel G prepared in the normal manner (19) were employed for all thinlayer studies. Concentrations and aliquots of solutions were chosen such that 3.0 pg. of DNP-amine was spotted on the plate. The solvent, hexane: ether, 70:30, was poured into the clean, dry battery jar just prior to running the chromatogram. Pre-saturation of the chamber must be avoided. The plates were run two times with the same solvent system, but fresh solvent was used each time. After the chromatograms were run, the plates were air-dried and marked into zones as shown in Figure 1. The adsorbent in the area of interest was loosened and removed by suction into a disposable capillary pipet. The adsorbent was then eluted with chloroform, which was evaporated to dryness with a stream of air. The residue was dissolved in a measured volume of benzene, usually 1.0 ml., and the amount present was determined by gas chromatography.
Reagents. The borate buffer was a 2.5y0 aqueous solution of NapBa07. 10H20. The DNFB reagent was prepared by dissolving 0.75 ml. of 2,4dinitrofluorobenzene (Eastman) in spectroquality p-dioxane (Rlatheson, Cole-
-
7-
man, and Bell). Cyclohexane was purified by pouring 500 ml. through a 40-gram column of silica gel, 0.2-0.5 mm . General Procedure. The procedure described is for an aqueous solution containing 1.0 Mg./ml. of each amine, either as the free base or a salt of the amine and is a modification of Kolbenzen's method (12). Ten milliliters of sample and/or standard solution is pipetted into a 50-ml. conical flask. Five milliliters of borate buffer and 2 ml. of DNFB reagent are added to the flask which is then placed in a 60" C. water bath for
n
/I
MINUTES
Figure 5.
Gas chromatogram of the Zone II standards See Table I for peak identification
VOL. 38, NO. 8, JULY 1966
1055
Table 111.
Gas Chromatographic Data for DNP-Amines
Sensitivity Retention Relative (mole/sec.) No. Amine (min.) retentiona x 10-16 11 Diethyl 6.42 1.00 3.3 8 t-Butyl 7.65 1.19 4.8 4 Isopropyl 8.78 1.37 4.2 Methylethyl 10 8.98 1.40 5.0 6 sec-Butyl 9.97 1.55 4.6 Dimethyl 9 10.62 1.66 5.3 7 Isobutyl 12.03 1.88 5.4 2 Ethyl 12.57 1.92 6.7 3 n-Propyl 13.39 2.09 5.7 5 n-Butyl 16.22 2.53 5.5 Methyl 1 16.44 2.60 7.9 a The ratio of the retention of the compound to that of Compound No. 11.
Table IV.
R, Values of DNP-Amines Compound no. Av. R, 0.46 0.83 1.12 1.16 1.34 1.38 1.41 1.47 8 0.41 9 0.69 10 1.00 I1
20 minutes. Two milliliters of 2N NaOH is added and the solution is heated an additional 30 minutes. It is then cooled and quantitatively transferred to a 125-ml. separatory funnel with deionized water. Extraction with 10.0 ml. of purified cyclohexane is carried out. After the layers separate, the aqueous layer is discarded and the cyclohexane is washed with three 15-ml. portions of 0.lN KasCOl. The cyclohexane is poured into a small flask or vial containing 1 to 2 grams of anhydrous Xa2S04. This solution may be analyzed directly by GLPC if amines are not present which interfere with each other. If separation by TLC is required, 5.0 ml. of the cyclohexane solution is pipetted into a vial, evaporated to dryness, 0.1 ml. of benzene is added to the residue, and 20 pl. is spotted on a thin-layer plate. The procedure described above is then followed. RESULTS AND DISCUSSION
Gas Chromatography. A number of different column packings were tested during the course of this investigation. The most satisfactory separations were obtained with ECKSS-?If or DEGS as the liquid phase and the best shaped peaks with Gas Chrom Q as the solid support. The DEGS was chosen because of its slightly better stability a t the operating temperatures required. These 2yo columns usually yielded satisfactory results for 2 to 3 weeks. Complete separation of all of the amine derivatives was not possible with the columns studied if retention 1056
ANALYTICAL CHEMISTRY
times were to be kept reasonably short. Figure 2 is a gas chromatogram of a solution containing the 11 DSP-amines a t a concentration of 3.0 kg./ml. each. Retention data are presented in Table 111 with relative retentions based on that of N,N-diethyl-2,4-dinitroaniline (Compound No. 11). The extent of branching in the aliphatic chain is apparently the dominating influence on retention. As can be seen in Figure 3, the four butylamines are nicely resolved but the order of their elution from the column is not related to their melting points. However, the more highly branched compounds exhibit the shorter retention times. The same holds true for the two propylamines. To some extent, though, reten-
tion increases with increasing chain length. Methylamine is an exception to this statement although it does hold true for the C2-Cs amines. (The retention time of the DNP derivative of n-amylamine is 21.1 minutes under the conditions described.) The range of the sensitivity values listed in Table I11 is rather small and can probably be considered to be essentially equal. This is not unexpected, however, since the detector should respond only to the aromatic nitro group and the nature of the alkyl group should not affect the detectability of the compounds by the electron affinity detector. Thin-Layer Chromatography. Of the several solvent systems tested, hexane :ether (70: 30) yielded the best separation of the DNP-amines. This system is very similar to that utilized by illangold (16). The separations obtained are illustrated in Figures 1 and 4. Movement on the plate is dependent on the mass of the alkyl groups and not on the extent of branching. The R. values reported in Table IV are calculated from the Rf values using Compound No. 11 as the reference standard, The compounds located in each of the zones shown in Figure 1 are completely resolved by gas chromatography. This is illustrated in Figures 3, 5 , and 6 where gas chromatograms of mixed standards from each of the zones are presented. Good resolution and nearly
10-
9-
8-
7W
6-
Ln
z $5W
a: 43-
21-
MINUTES
Figure 6.
Gas chromatogram of the Zone I standards See Table I for peak identification
Table V. Recoveries of Amines through Dinitrophenylation Procedure
Compound no. 1
2 3 4 5 6
7 8 9
10 11
Recoveries (70) 7.6 7.6 62.8 65.9 96.0 103.0 74.6 88.6 102.0 85.5 82.5 87.5 79.6 81.4 39.9 49.6 31.8 34.0 72.6 72.6 68.8 68.8
Average 7.6 64.4 99.5 81.6 93.8 85.0 80.5 44.8 32.9 72.6 68.8
base line separations are obtained for all of the compounds which permits reliable quantitation. Dinitrophenylation. The dinitrophenylation procedure is not quantitative for all of the amines. I n one experiment, duplicate aliquots of 1.0pg./ml. solutions of each of the amines were dinitrophenylated and assayed by gas chromatography without prior thinlayer chromatography. The recoveries observed when compared to direct standard solutions are listed in Table V. Other authors (8, 12) have noted variability in the reactivity of DNFB with amines. However, the procedure can still be used for quantitative analysis as is shown by the analytical curves plotted in Figure 7 . Corrections are made in this figure for solutions which required a greater volume of cyclohexane than that specified in the procedure. The optimum concentrations of amines were obviously not chosen to obtain these curves but they were sufficient to demonstrate the linearity of response with concentration. The gas chromatogram of the reagent blank exhibits several extraneous peaks when thin-layer chroniatography is not employed. These occur within the first 2 minutes and are of no consequence in the determination of amines. Also, the benzene solutions of the residues from eluates of blank portions of the thin-layer plates show one or more noninterfering peaks. No attempt has been made to identify any of these peaks. LITERATURE CITED
(1) Arad-Talmi, Y., Levy, J., T’ofsi, D.. ,J. Chromntoa. 13. ,565 (1964).
Chem 27, 452 (l!
COM POUN D 6
40
35
9
30
25
2
20
15
5
10
5
1
/ A
C Figure 7.
+
0.5
I
1.o
I
I
1.5
2.0
Plot of peak height vs. amine concentration for selected amines See Table I for compound identification
(6) Brauniger, H., Spangenberg, K., Pharmazie 12. 335 (1957): C.A. 54. 22668~
(1960). (7) Burks, R. E., Jr., Baker, E. B., Clark, P., Esslinger, J., Lacy, J. C., Jr., J . Agr. Food Chem. 7, 778 (1959). (8) Dubin, D. T., J . Biol. Chem. 235, 783 (1960). (9) Graymore, J., J . Chem. SOC.1938, 3 “1,
1611.
(10) Hollingsworth, B. L., Zbid., 1959, 2420. (11) Kolbezen, hI. J., University of California, Riverside, Calif., Private communication, September 1964. (12) Kolbezen, M. J., Eckert, J. W., Bretschneider, B. F., ANAL.CHEM.34, 583 (1962). (13) Landault, C., Guiochon, G., J . Chromatog. 13, 327 (1964). (14) Landowne, R. A., Lipsky, S. R., ANAL.CHEV.34, 762 (1962). (15) Landowne, R. A., Liuskv. S. R.. Nature 199, 141 (1963). - ” ’ (16) Mangold, H. ,K. in “Thin Layer Chromatography, E. Stahl, ed., p.
167, Academic Press, New York, 1963. (17) McIntire, F. C., Clements, L. M., Sproull, M,, ANAL. CHEM. 25, 1757 f1953). (18)-O’Donnel, J. F., Mann, C. K., Zbid., 36, 2097 (1964). (19) Stahl, E., Chemiker Ztg. 82, 323 (1958). (20) Stahl. E. in “Dunnschicht-chroma’ toeraDhie.” E. Stahl. ed.. A. 310. SGinger-qerlag, Berlin: 1962: (21) Sze, Y. L., Borke, M.L., Ottenstein, D. bl., ANAL.CHEM.35, 240 (1963). (22) Vanden Heuvel, W. J. A., Gardiner, W. L., Hornina. -, E. C.. Zbid.. 36. 1550 (1964j. (23) Vizgert, R. V., Zhur. Obshchei Khim. 30, 3438 (1960); C.A. 55, 19846d r
I
- - - 7
,
flQ61). \ _ _ _ _
(24) Whk, S., Gitlow, S. E., Franklin, M. J., Carr, H. E., Clin. Chin. Acta 10, 193 (1964). RECEIVED for review March 9, 1966. Accepted April 15, 1966.
VOL. 38, NO. 8, JULY 1966
1057
