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Some observations on thin-layer chromatography for identification and separation of amino compounds on mixed adsorbents with benzene-containing eluents
MICROCHEMICAL
42, 206-217
JOURNAL
(1990)
Some Observations on Thin-Layer Chromatography for Identification and Separation of Amino Compounds on Mixed Adsorbents with Benzene-Containing Eluents M. Department
A.
AJMAL,’
MOHAMMAD,
AND
S. ANWAR
of Applied Chemistry, Z. H. College of Engineering Aligarh Muslim University, Aligarh 202002 (U.P.), Received
and Technology, India
January 5, 1990; accepted February 5, 1990
Thin-layer chromatography of 14 amino compounds on mixed adsorbents containing silica gel and alumina or cellulose has been performed with mixed organic eluents containing benzene. The results obtained on plain silica gel, alumina, and cellulose have been compared with those obtained on mixed adsorbents. The effect of metal salts on the separation of o-nitroaniline from p-nitroaniline has been investigated. Microgram quantities of diphenylamine and m- or p-phenylenediamine have been separated from one another. The proposed method is rapid and applicable to the identification and separation of amines over a wide range of concentration. 0 1990 Academic Press, Inc.
INTRODUCTION
Thin-layer chromatography (TLC) combined with modern layers, instrumentation, and optimized procedures continues to be a widely used technique for separating inorganics, organometallics, and organics. As an aid to pharmacists, TLC is being used in the biomedical and pharmaceutical fields. The majority of TLC work has been performed on silica gel layers, followed by chemically bonded reversed-phase layers, cellulose, ion exchangers, layers impregnated with complexing reagents, and alumina with organic or organic-aqueous mobile phases. Because of the physiological importance of amines, improved methods are continually being developed to analyze them (Z-7). Papers impregnated with Cu(I1) sorbed onto zinc silicate were used to investigate ligand exchange of amines (8). A TLC method for the detection of aminophenols and aromatic amines on NaNO,-impregnated silica gel layers has been developed (9). In order to achieve desired and improved separation of amines, several workers have tried HPLC, TLC, and PC (5, 10-14). The inorganic salts of the alkali metals are frequently used in organic synthesis and their presence in the final product may affect the chromatographic performance of organic substances. It is, therefore, worthwhile to study the effect of alkali metal salts on the separation of organic substances. The present study was undertaken in order to develop a simple, rapid, and reliable TLC method for the identification and separation of aromatic amines in the presence of metal salts. An attempt has also been made to separate amines at micro-
r To whom correspondence
should be addressed. 206
0026-265X/90 $1 SO Copyright 0 19!Xl by Academic Press, Inc. All rights of reprcduct~on in any form reserved.
TLC
FOR
IDENTIFICATION
OF
AMINO
207
COMPOUNDS
gram and milligram levels. Some new sorbent phases capable of causing differential migration of amines have been identified. EXPERIMENTAL Apparatus
A TLC apparatus (Toshniwal, India) was used to prepare silica gel layers (0.25 mm) on 20 x 3-cm glass plates. Glass jars (29 x 6 cm) were used for the development of chromatographic plates. A glass sprayer was used to spray the reagent over the plates to detect the spots. Reagents
Silica gel G and methanol were from Qualigene, India; alumina were from CDH, India; benzene (BDH, India) and cyclohexane were used. All other reagents were of analar grade. Amines
and cellulose (SDH, India)
Studied
Aniline (AL), diphenylamine (DPA), p-nitroaniline (p-NAL), o-nitroaniline (oNAL), o-chloroaniline (o-CAL), p-chloroaniline (p-CAL), I-naphthylamine (NPA), p-toludine (p-TD), N,N-dimethylaniline (DMAL), m-phenylenediamine (m-PDA), p-phenylenediamine (p-PDA), and carbazole (CZ) were studied. One percent solutions of all amines in methanol were used as test solutions. Detection
A 1% alcoholic solution of p-dimethylaminobenzaldehyde containing 15-20 ml of concentrated H,SO, was used to detect all the amines except carbazole which was detected by spraying 1 .O M aqueous NaNO, solution on the chromatographic plates followed by concentrated H,SO,. The appearance of a dark green spot shows the presence of CZ. Solvent Systems
The following S, S, S, S, S, S,
solvent systems were used:
cyclohexane-benzene (5:2, 2:5, 3:4, 4:3, 0:7, 7:0, and 1:1, v/v) ethyl acetate-benzene (5:2, 2:5, 3:4, 4:3, 0:7, 7:0, v/v) acetic acid-benzene (2: 1, 1: 1, 2:5, v/v) acetone-benzene (1: 1, 1:2, 2: 1, v/v) carbon tetrachloride-benzene (1:2, 1: 1, 2: 1, v/v) ethyl methyl ketone-benzene (1:2, 1:1, 2:1, v/v).
Preparation
of TLC Plates
(a) Plain silica gel thin-layer plates. The silica gel plates were prepared by mixing silica gel with water in a 1:3 ratio with constant shaking until a homogeneous slurry was obtained. The resultant slurry was applied to the glass plates
208
AJMAL,
MOHAMMAD,
AND
ANWAR
with the help of an applicator to give a 0.25mm-thick layer. The plates were dried at room temperature and then activated at 100 + 5°C by heating in an electrically controlled oven for 1 h. The activated plates were stored in a closed chamber at room temperature until used. (b) Mixed silica gel-alumina or -cellulose plates. The synthetic mixtures containing alumina or cellulose and silica gel G in different ratios (1: 1, 2: 1, 1:2, w/w) were slurried with distilled water in a 1:3 ratio by shaking for 5-10 min. Thin layers of the resultant slurry were prepared by following the procedure as described in (a). Procedure
About 0.05 ml of test solution was spotted on a thin-layer plate with the help of a micropipet. The plates were developed in the chosen solvent system by the ascending technique. The solvent ascent was fixed to 10 cm in all cases. After development was complete, the plates were withdrawn from glass jars and dried at room temperature. The amines were detected and their R, (RF of leading front) and R, (RF of tailing front) values were determined. To study the effect of the presence of alkali salts on the separation of p-NAL from o-NAL, the synthetic mixtures were prepared by adding various concentrations of 1% acidified salt solutions to the synthetic mixture containing o-NAL and p-NAL in a 1: 1 ratio. The resultant mixture was spotted on the chromatoplate and the spot was allowed to dry. The plates were developed, the spots were detected, and the RF values of the separated amines were determined from their R, and R, values. The limits of detection of various amines were determined by spotting different amounts of amines on the chromatoplates, developing the plates, and detecting the spots. The method was repeated with successive lowering of the amount of amine until no spots were detected. The lowest amount of amine detected on the TLC plates was taken as the limit of detection. The effect of the amount of amine loaded on the RF value was studied by spotting a fixed volume (0.05 ml) of a standard solution of the amine concerned (l-10%) in methanol on the chromatoplate. The plates were developed and the spots were detected. The R, - RT values for each spot were determined. For the separation of a fixed amount of an amine from various amounts of another amine, synthetic mixtures of amines were prepared by adding various volumes of standard solution (10%) of the amine (A) into 0.05 ml of 1% amine (B). The resultant mixture was loaded on the chromatoplate. The plates were developed, the spots were detected, and the RF values of the separated amines were determined. RESULTS
AND DISCUSSION
The results obtained have been summarized in Figs. l-4 and Tables 14. In order to identify the possibilities of certain desired separations of amino compounds, various combinations of stationary and mobile phases were tried. All, 9 stationary
TLC
FOR
IDENTIFICATION
OF
AMINO
COMPOUNDS
209
phases (pure and mixed) and 48 mobile phases containing benzene were used. The development time for a lo-cm run ranged from 15 to 30 min, depending on the compositions of the mobile and the stationary phases. Among the mobile phases used, cyclohexane-benzene was found to be the best solvent system in terms of compactness of spots and clearness of detection. The chromatographic behavior of aromatic amines on silica gel layers in solvent systems containing variable concentrations of benzene and cyclohexane, acetic acid, formic acid, ethyl acetate, ethyl methyl ketone, or carbon tetrachloride is shown in Fig. 1. The following trends are noticeable in Fig. 1. 1. The plots shown in Fig. la pass through maxima and minima showing the significant effect of the proportional composition of the constituents of the mobile phase on the R, values of the amines. Thus, the cyclohexane-benzene system with variable proportions of constituting components can be used for the desired separations of amines. 2. The ethyl acetate-benzene system is useful for the selective separation of o-NAL (Fig. lb). 3. In the carbon tetrachloride-benzene (1:l) system, all amines except DPA, CZ, and DMAL showed maximum R, values. The R, values of DPA, CZ, and DMAL increase with the increase in carbon tetrachloride concentration in the mobile phase (Fig. lc). It seems that both basicity and steric effect influence the R, value of the amines. 4. The R, value of AL decreases sharply with the increase in acetic acid concentration or the decrease in benzene concentration. However, the R, values for CZ, DPA, and p-NAL showed gradual decreases. The R, value of chloroaniline remained constant (RF = 1 .OO) at all concentrations of acetic acid or benzene (Fig. Id). m-PDA showed the maximum R, value (RF = 1.0) followed by a sharp decrease as soon as the acetic acid concentration was increased beyond 50%. All other amines show the minimum R, value in the acetic acid-benzene (1: 1) system. 5. In the ethyl methyl ketone-benzene system most amines showed constant R, values at all concentrations of benzene in the mobile phase (Fig. le). However, the R, values for p-TD, m-PDA, and p-NAL showed a gradual increase with the decrease in benzene concentration. 6. Analogous to the ethyl methyl ketone-benzene system, the formic acidbenzene system was found to be of limited practical use for the separation of amines because of the small variation in the RF values of amines with the increase in concentration of formic acid (Fig. If). 7. Comparison of Fig. Id and If showed that the R, values of amines are influenced differently by formic acid and acetic acid. The sorption of amines on cellulose, alumina, or silica gel layers with cyclohexane-benzene (1: 1) has been summarized in Fig. 2. On cellulose layers, m- and p-PDA were strongly adsorbed and remained at the point of application while all other amines moved with the solvent front. Thus, diamines can be well separated from monoamines on cellulose layers using cyclohexane-benzene (1: 1) as the mobile phase. All amines except DPA and CZ were retained on silica gel layers and gave very low R, values. DPA and CZ moved with the solvent front. Most of the amines showed higher mobility on alumina than on silica gel in the
210
AJMAL,
MOHAMMAD,
AND
ANWAR
I > . >
TLC
FOR IDENTIFICATION
OF AMINO
COMPOUNDS
211
4--jJlti---
3
212
AJMAL,
MOHAMMAD,
AND
ANWAR
FIG. 2. Sorption of amines on cellulose, alumina, and silica gel layers with the cyclohexanebenzene (l:l, v/v) solvent system. 1, m-PDA; 2, p-PDA; 3, p-NAL; 4, m-NAL; 5, I-NPA; 6, m-CAL; 7, AL; 8, DPA; 9, CZ; 10, p-CAL; 11, p-TD; 12, NJ,-DMAL.
cyclohexane-benzene (1: 1) solvent system. The lower RF values on silica gel layers may be attributed to the interaction between the acid surface group of the silica gel and the aromatic amines. This interaction may lead to the existence of
It is well known that hydrogen bonding plays a crucial role in binding the carcinogen to nucleic acid analogues of DNA (15). It is interesting to note from Fig. 2 that amines containing two aryl groups, i.e., DPA or CZ (RF = 0.95), can be selectively separated from amines containing one aryl group like aniline, substituted aniline, and aniline derivatives (RF = 0.0-0.35) on silica gel layers. The higher R, values for CZ and DPA may be attributed to their lower basicity. It is clear from Fig. 2 that most of the amines showed different selectivities toward the three adsorbents described above. Therefore, the retention behavior of amines on mixed adsorbents containing silica gel and alumina or cellulose is amenable to study. The R, values of amines in cyclohexane-benzene (1: 1) are illustrated in Fig. 3 as a function of the percentage concentration of silica gel in the mixture containing silica gel and alumina or cellulose as sorbent phase. It is evident from Fig. 3 that there are several possible selective separations of amines on mixed sorbent phases, for example, p-CAL from o-CAL on silica gel-alumina and silica gel-cellulose containing 33 and 50% silica gel, respectively. Similarly, silica gel-alumina containing 33% silica gel may be used to separate DPA from p-TD or o-NAL. Silica gel-cellulose containing 66% silica gel was found to be a good adsorbent for selective separation of DPA.
TLC FOR IDENTIFICATION
33 Concentrot~on
SilCO
of
gel
(*A)
50
66
33
of
concentration
S~l~CO gel
OF AMINO
(‘/.I
50
33
66
Concentrot~on SIIICO
213
COMPOUNDS
gel
of (%I
50
66
Concentrotlon Sil~CO
of
gelf%)
FIG. 3. RFvalues of amines as a function of the silica gel concentration in cyclohexane-benzene (1: 1, v/v). 0, Silica gel-alumina adsorbent; 0 silica gel-cellulose adsorbent.
Separations on TLC are sometimes hampered due to the appearance of tailed spots caused by high loading of sample. In order to determine the maximum amount of an amine which can be separated without tailing on silica gel-cellulose (2:l) with cyclohexane-benzene (I:l), we calculated the R, and RT values for variable concentrations of amines. Figure 4 indicates that most of the amines produce compact spots (RL - R, < 0.3) in the l-IO% concentration range (loading amount l-10 mg) of sample. p-NAL at 10% concentration and aniline at all concentrations (l-10%) gave tailed spots. Table 1 indicates that the proposed method for the identification of amines on TLC plates is reasonably sensitive. Aniline down to 0.05 u,g can be detected on TLC plates. The qualitative separations achieved on mixed adsorbent layers containing silica gel and alumina or cellulose with various solvent systems are summarized in Table 2. In addition to these separations, AL from m- or p-PDA, p-CAL, DPA from m- or p-PDA, p-NAL from NPA, m-PDA or p-PDA, p-CAL, and o-CAL from m- or p-PDA were achieved on plain silica gel layers in different solvent systems. The results for the separation of DPA from m-PDA or p-PDA at milligram levels are tabulated in Table 3: 500 t.r,gof DPA can be well separated from 10
214
AJMAL,
MOHAMMAD,
AND
ANWAR
Loodhg
,O’,. 5% 25% Loodmg amount
FIG.
(l:l,
3% lmg)
10% 5% 2.5’1. Loading amount
IV. (mg)
amount
10% 5% 2.5% I% Loodmg amount (mgl
4. Effect of loading amount of amines on the R, values. Mobile phase, cyclohexane-benzene v/v); stationary phase, silica gel-cellulose (2:l, w/w). 0, R, values; 0, R, values.
mg of p-PDA concentration or m-PDA is separation of
and vice versa. The mutual separation range 500 pg-5 mg is possible. However, loaded, the spots are barely separated. o-NAL from p-NAL is not hampered
of DPA and m-PDA in the when 10 mg of either DPA Table 4 indicates that the by the presence of certain
TABLE 1 Limit of Detection of Amines on Silica Gel-Cellulose (2: 1) Layers with the Cyclohexane-Benzene (1: 1) Solvent System No.
Amines
Limit of detection w
1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13.
Aniline Diphenylamine p-Nitroaniline I-Naphthylamine o-Chloroaniline o-Nitroaniline m-Phenylenediamine p-Phenylenediamine p-Chloroaniline p-Toludine Carbazole m-Nitroaniline m-Chloroaniline
5.0 x 10-a 5.0 x 10-t 5.0 x 10-a 5.0 5.0 5.0 x 10-t 5.0 x 10-t 5.0 x 10-t 5.0 x 10-t 5.0 x 10-t 1.0 x 10-l 5.0 x 10-l 5.00
(mg I
TLC
Separation Stationary
FOR
of Amines
on Mixed
phase
Mobile
Silica gel G-AI,O, 2:1 2:1
1. 2.
IDENTIFICATION
OF
TABLE Adsorbent
AMINO
2 Layers
phase
with
Various
DPA (10.0 - 9.$po-NAL (7.0 - 5.0)
Benzene
o-CAL
(8.5 - 6.5)
4.
1:l
Benzene
o-CAL
(8.5 - 6.5)
5.
1:l
Benzene
o-NAL
(7.0 - 5.0)
6.
1:2
Benzene
DPA (9.0 - 8.0)
7.
I:2
Benzene
o-CAL
8.
2:1
Cyclohexone-benzene
(1: 1)
DPA (8.0 - 6.0)
9.
2:1
Cyclohexane-benzene
(1: 1)
p-NPA
(4.5 - 3.0)
10.
I:2
Cyclohexane-benzene
(1:l)
o-CAL
(7.5 - 5.0)
11.
I:2
Cyclohexane-benzene
(1:l)
DPA (8.0 ~ 7.0)
12.
1:l
Cyclohexane-benzene
(1: 1)
o-CAL
(2: 1) ( 1: 1) ( 1:2) ( 1: 1) (2: 1)
p-NAL (3.5 DPA (6.5 p-NAL (1.5 p-NAL (1.5 p-NAL (0.0 p-NAL (1.5 DPA (3.0 -
Separation
of DPA
Benzene Benzene Cyclohexane-benzene Cyclohexane-benzene Cyclohexene-benzene Cyclohexane-benzene Cyclohexane-benzene
TABLE m- and p-PDA at Milligram with the Cyclohexane-Benzene
from
Amount No.
Separation DPA*-p-PDA DPA*-p-PDA DPA*-p-PDA DPA*-p-PDA DPA*-m-PDA DPA*-m-PDA DPA*-m-PDA p-PDA-DPA p-PDA*-DPA p-PDA*-DPA m-PDA*-DPA m-PDA*-DPA m-PDA*-DPA
* Refers
to the amount
of corresponding
(9.0 ~ 7.5)
(7.0 - 5.0)
Levels
or m-PDA (0.5 - 0.0) -m-PDA (1.0 - 0.0) -p-PDA (0.5 - 0.0) -in-PDA (0.5 - 0.0) -p-PDA (1.0 - 0.0) -m-PDA (0.3 - 0.0) -p-PDA (1.0 - 0.0) -m-PDA (1.0 - 0.0) -p-PDA (1.0 - 0.0) -m-PDA (0.0 0.0) -p-PDA (0.0 - 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 ~ 0.0) -p-NAL (0.5 - 0.0) -m-PDA (0.5 - 0.0) -p-PDA (0.5 - 0.0) -p-NAL (0.10 - 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 - 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 - 0.0) -m-PDA (0.4 - 0.0) -p-PDA (0.3 - 0.0) -p-NAL (0.0 ~ 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 - 0.0) -o-NAL (7.0 - 6.0) -cz (10.0 - 9.5) -o-NAL (4.5 - 3.0) -o-NAL (5.5 - 3.0) -o-NAL (4.0 - 3.0) -o-NAL (5.0 - 3.0) -cZ (9.0 - 8.0)
on Silica Gel-Cellulose (I : 1) System
(2: 1) Layers
loaded
0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5*
10 (RL 1 5 10 12 1 5 10 1 5 10 1 5 IO
-
- 2.0) 3.5) ~ 1.0) ~ 0.0) - 0.0) - 0.5) 2.0)
- &I
3
(mg)
R,
Phases 10 CR,
Benzene Benzene
2:l
Silica-cellulose 1:l 1:2 1:l 1:2 1:2 2:1 1:2
Mobile
Separations
3.
1. 2. 3. 4. 5. 6. I.
215
COMPOUNDS
R,- values
for DPA.
- RJ
(IO - 8.5)*, (2.5 - 0) (9.8 - 8.6)*, (4.0 - 0) (10 - 7.5)*, (5.5 - 0) (IO - 7.8)*, (6.0 - 0) (IO - 9)*, (3.0 - 0) (IO - 9)*, (4.0 - 0) (IO - 9)*. (9 - 0) (1.5 - O)*, (9.8 - 9.0) (6 - O)*, (10 - 6.5) (2.5 - O)*, (9.6 - 8.6) (3.0 - o)*, (IO - 9) (5.0 - o)*, (IO - 9) (7.0 - oj*, (10 - 7)
216
AJMAL,
MOHAMMAD, TABLE
AND
ANWAR
4
Separation of o-NAL from p-NAL in the Presence of Metal Salts on Silica Gel-Cellulose Layers in the Cyclohexane-Benzene (1: 1) System No.
Metal salt
1.
Sodium chloride
2.
Copper sulfate
3.
Ammonium chloride
4.
Nickle chloride
5.
Cobalt sulfate
6.
Chrominum chloride
I.
Manganese chloride
8.
Vanadium sulfate
Amount added (mg) 10.0 30.0 45.0 10.0 30.0 50.0 10.0 30.0 50.0 10.0 30.0 50.0 10.0 30.0 10.0 30.0 40.0 10.0 30.0 50.0 10.0 30.0 40.0
a RL - RT values for the separation of o-NAL and p-NAL o-NAL (3.8 - 2.8) andp-NAL (1.5 - 0.5).
p-NAL” 10 (RL - JW (0.5 (0.8 (0.8 (0.5 (0.8 (0.5 (0.5 (0.8 (0.5 (0.5 (0.8 (0.6 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5
-
0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
(2: 1)
o-NAL” 10 (RL - &) (4.0 (4.0 (3.1 (3.5 (4.0 (2.8 (3.0 (4.2 (2.6 (3.0 (4.8 (3.0 (3.0 (3.0 (3.0 (3.3 (3.0 (4.0 (3.5 (2.5 (2.6 (2.6 (2.8
-
3) 3.0) 1.6) 2.5) 3.0) 2.0) 2.0 3.2) 2.0) 2.0) 3.8) 2.0) 2.2) 2.2) 2.0) 2.2) 2.0) 3.0) 2.5) 1.4) 2.0) 2.0) 1.8)
in the absence of salt are as follows:
metal salts up to 45-50 mg. However, the tolerance of cobalt sulfate is only up to 30 mg. The amines cannot be detected when the loading of metal salts exceeds the limiting value noted in Table 4. An increase in resolution was observed in the presence of metal salts compared to the separation of o-NAL from p-NAL in the absence of metal salts from the synthetic mixture. ACKNOWLEDGMENTS The authors thank Professor K. T. Nasim for providing research facilities. S.A. thanks the U.G.C. for financial assistance.
REFERENCES 1.
2. 3. 4. 5. 6.
Nabi, S. A.; Moharnmad, A.; Qureshi, P. M. Talanta, 1979, 26, 1179. Qureshi, M.; Nabi, S. A.; Khan, I. A.; Qureshi, P. M. Talanta, 1982, 29, 757. Papadoyannics, I. N. Microchem. J., 1985, 32, 220. Cserhati, T.; Bordes, B.; Szogyi, M. J. Chromatogr. Sci., 1986, 24, 302. Khulbe, K. C.; Mann, R. S. Fresenius Z. Anal. Chem., 1988, 330, 642. Gennaro, M. C.; Marengo, E. Chromatographia, 1988, 25, 603.
TLC FOR IDENTIFICATION
OF AMINO COMPOUNDS
7. Iskander, M. L.; Medien, H. A. A.; Nashad, S. Microchem. J., 1987, 36, 368. 8. Singh, D. K.; Darbari, A. Chromatographia, 1987, 23, 93. 9. Dhillan, R. S.; Singh, J.; Gautam, V. K.; Chhabra B. R. J. Chromarogr., 1988, 435, 256. 10. Kuhn, A. 0.; Lederer, M. J. Chromatogr., 1988, 440, 165. 11. Geerdink, R. B. J. Chromatogr., 1988, 44.5, 237. 12. Phars, D. Y.; Laiden, P. C.; Siggia, S. Chromatogr. Sci., 1985, 23(9), 391-396. 13. Hovea, N.; Elena, M.; Teodar, H. Rev. Chim., 1985, 36, 446. 14. Dimov, N.; Agapona, N.; Nauchnoizseled, Tr. Khim Farm. Inst., 1986, 16, 113. IS. Hathway, D. E.; Kolar, G. F. Chem. Sot. Rev., 1980, 241.
217
42, 206-217
JOURNAL
(1990)
Some Observations on Thin-Layer Chromatography for Identification and Separation of Amino Compounds on Mixed Adsorbents with Benzene-Containing Eluents M. Department
A.
AJMAL,’
MOHAMMAD,
AND
S. ANWAR
of Applied Chemistry, Z. H. College of Engineering Aligarh Muslim University, Aligarh 202002 (U.P.), Received
and Technology, India
January 5, 1990; accepted February 5, 1990
Thin-layer chromatography of 14 amino compounds on mixed adsorbents containing silica gel and alumina or cellulose has been performed with mixed organic eluents containing benzene. The results obtained on plain silica gel, alumina, and cellulose have been compared with those obtained on mixed adsorbents. The effect of metal salts on the separation of o-nitroaniline from p-nitroaniline has been investigated. Microgram quantities of diphenylamine and m- or p-phenylenediamine have been separated from one another. The proposed method is rapid and applicable to the identification and separation of amines over a wide range of concentration. 0 1990 Academic Press, Inc.
INTRODUCTION
Thin-layer chromatography (TLC) combined with modern layers, instrumentation, and optimized procedures continues to be a widely used technique for separating inorganics, organometallics, and organics. As an aid to pharmacists, TLC is being used in the biomedical and pharmaceutical fields. The majority of TLC work has been performed on silica gel layers, followed by chemically bonded reversed-phase layers, cellulose, ion exchangers, layers impregnated with complexing reagents, and alumina with organic or organic-aqueous mobile phases. Because of the physiological importance of amines, improved methods are continually being developed to analyze them (Z-7). Papers impregnated with Cu(I1) sorbed onto zinc silicate were used to investigate ligand exchange of amines (8). A TLC method for the detection of aminophenols and aromatic amines on NaNO,-impregnated silica gel layers has been developed (9). In order to achieve desired and improved separation of amines, several workers have tried HPLC, TLC, and PC (5, 10-14). The inorganic salts of the alkali metals are frequently used in organic synthesis and their presence in the final product may affect the chromatographic performance of organic substances. It is, therefore, worthwhile to study the effect of alkali metal salts on the separation of organic substances. The present study was undertaken in order to develop a simple, rapid, and reliable TLC method for the identification and separation of aromatic amines in the presence of metal salts. An attempt has also been made to separate amines at micro-
r To whom correspondence
should be addressed. 206
0026-265X/90 $1 SO Copyright 0 19!Xl by Academic Press, Inc. All rights of reprcduct~on in any form reserved.
TLC
FOR
IDENTIFICATION
OF
AMINO
207
COMPOUNDS
gram and milligram levels. Some new sorbent phases capable of causing differential migration of amines have been identified. EXPERIMENTAL Apparatus
A TLC apparatus (Toshniwal, India) was used to prepare silica gel layers (0.25 mm) on 20 x 3-cm glass plates. Glass jars (29 x 6 cm) were used for the development of chromatographic plates. A glass sprayer was used to spray the reagent over the plates to detect the spots. Reagents
Silica gel G and methanol were from Qualigene, India; alumina were from CDH, India; benzene (BDH, India) and cyclohexane were used. All other reagents were of analar grade. Amines
and cellulose (SDH, India)
Studied
Aniline (AL), diphenylamine (DPA), p-nitroaniline (p-NAL), o-nitroaniline (oNAL), o-chloroaniline (o-CAL), p-chloroaniline (p-CAL), I-naphthylamine (NPA), p-toludine (p-TD), N,N-dimethylaniline (DMAL), m-phenylenediamine (m-PDA), p-phenylenediamine (p-PDA), and carbazole (CZ) were studied. One percent solutions of all amines in methanol were used as test solutions. Detection
A 1% alcoholic solution of p-dimethylaminobenzaldehyde containing 15-20 ml of concentrated H,SO, was used to detect all the amines except carbazole which was detected by spraying 1 .O M aqueous NaNO, solution on the chromatographic plates followed by concentrated H,SO,. The appearance of a dark green spot shows the presence of CZ. Solvent Systems
The following S, S, S, S, S, S,
solvent systems were used:
cyclohexane-benzene (5:2, 2:5, 3:4, 4:3, 0:7, 7:0, and 1:1, v/v) ethyl acetate-benzene (5:2, 2:5, 3:4, 4:3, 0:7, 7:0, v/v) acetic acid-benzene (2: 1, 1: 1, 2:5, v/v) acetone-benzene (1: 1, 1:2, 2: 1, v/v) carbon tetrachloride-benzene (1:2, 1: 1, 2: 1, v/v) ethyl methyl ketone-benzene (1:2, 1:1, 2:1, v/v).
Preparation
of TLC Plates
(a) Plain silica gel thin-layer plates. The silica gel plates were prepared by mixing silica gel with water in a 1:3 ratio with constant shaking until a homogeneous slurry was obtained. The resultant slurry was applied to the glass plates
208
AJMAL,
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with the help of an applicator to give a 0.25mm-thick layer. The plates were dried at room temperature and then activated at 100 + 5°C by heating in an electrically controlled oven for 1 h. The activated plates were stored in a closed chamber at room temperature until used. (b) Mixed silica gel-alumina or -cellulose plates. The synthetic mixtures containing alumina or cellulose and silica gel G in different ratios (1: 1, 2: 1, 1:2, w/w) were slurried with distilled water in a 1:3 ratio by shaking for 5-10 min. Thin layers of the resultant slurry were prepared by following the procedure as described in (a). Procedure
About 0.05 ml of test solution was spotted on a thin-layer plate with the help of a micropipet. The plates were developed in the chosen solvent system by the ascending technique. The solvent ascent was fixed to 10 cm in all cases. After development was complete, the plates were withdrawn from glass jars and dried at room temperature. The amines were detected and their R, (RF of leading front) and R, (RF of tailing front) values were determined. To study the effect of the presence of alkali salts on the separation of p-NAL from o-NAL, the synthetic mixtures were prepared by adding various concentrations of 1% acidified salt solutions to the synthetic mixture containing o-NAL and p-NAL in a 1: 1 ratio. The resultant mixture was spotted on the chromatoplate and the spot was allowed to dry. The plates were developed, the spots were detected, and the RF values of the separated amines were determined from their R, and R, values. The limits of detection of various amines were determined by spotting different amounts of amines on the chromatoplates, developing the plates, and detecting the spots. The method was repeated with successive lowering of the amount of amine until no spots were detected. The lowest amount of amine detected on the TLC plates was taken as the limit of detection. The effect of the amount of amine loaded on the RF value was studied by spotting a fixed volume (0.05 ml) of a standard solution of the amine concerned (l-10%) in methanol on the chromatoplate. The plates were developed and the spots were detected. The R, - RT values for each spot were determined. For the separation of a fixed amount of an amine from various amounts of another amine, synthetic mixtures of amines were prepared by adding various volumes of standard solution (10%) of the amine (A) into 0.05 ml of 1% amine (B). The resultant mixture was loaded on the chromatoplate. The plates were developed, the spots were detected, and the RF values of the separated amines were determined. RESULTS
AND DISCUSSION
The results obtained have been summarized in Figs. l-4 and Tables 14. In order to identify the possibilities of certain desired separations of amino compounds, various combinations of stationary and mobile phases were tried. All, 9 stationary
TLC
FOR
IDENTIFICATION
OF
AMINO
COMPOUNDS
209
phases (pure and mixed) and 48 mobile phases containing benzene were used. The development time for a lo-cm run ranged from 15 to 30 min, depending on the compositions of the mobile and the stationary phases. Among the mobile phases used, cyclohexane-benzene was found to be the best solvent system in terms of compactness of spots and clearness of detection. The chromatographic behavior of aromatic amines on silica gel layers in solvent systems containing variable concentrations of benzene and cyclohexane, acetic acid, formic acid, ethyl acetate, ethyl methyl ketone, or carbon tetrachloride is shown in Fig. 1. The following trends are noticeable in Fig. 1. 1. The plots shown in Fig. la pass through maxima and minima showing the significant effect of the proportional composition of the constituents of the mobile phase on the R, values of the amines. Thus, the cyclohexane-benzene system with variable proportions of constituting components can be used for the desired separations of amines. 2. The ethyl acetate-benzene system is useful for the selective separation of o-NAL (Fig. lb). 3. In the carbon tetrachloride-benzene (1:l) system, all amines except DPA, CZ, and DMAL showed maximum R, values. The R, values of DPA, CZ, and DMAL increase with the increase in carbon tetrachloride concentration in the mobile phase (Fig. lc). It seems that both basicity and steric effect influence the R, value of the amines. 4. The R, value of AL decreases sharply with the increase in acetic acid concentration or the decrease in benzene concentration. However, the R, values for CZ, DPA, and p-NAL showed gradual decreases. The R, value of chloroaniline remained constant (RF = 1 .OO) at all concentrations of acetic acid or benzene (Fig. Id). m-PDA showed the maximum R, value (RF = 1.0) followed by a sharp decrease as soon as the acetic acid concentration was increased beyond 50%. All other amines show the minimum R, value in the acetic acid-benzene (1: 1) system. 5. In the ethyl methyl ketone-benzene system most amines showed constant R, values at all concentrations of benzene in the mobile phase (Fig. le). However, the R, values for p-TD, m-PDA, and p-NAL showed a gradual increase with the decrease in benzene concentration. 6. Analogous to the ethyl methyl ketone-benzene system, the formic acidbenzene system was found to be of limited practical use for the separation of amines because of the small variation in the RF values of amines with the increase in concentration of formic acid (Fig. If). 7. Comparison of Fig. Id and If showed that the R, values of amines are influenced differently by formic acid and acetic acid. The sorption of amines on cellulose, alumina, or silica gel layers with cyclohexane-benzene (1: 1) has been summarized in Fig. 2. On cellulose layers, m- and p-PDA were strongly adsorbed and remained at the point of application while all other amines moved with the solvent front. Thus, diamines can be well separated from monoamines on cellulose layers using cyclohexane-benzene (1: 1) as the mobile phase. All amines except DPA and CZ were retained on silica gel layers and gave very low R, values. DPA and CZ moved with the solvent front. Most of the amines showed higher mobility on alumina than on silica gel in the
210
AJMAL,
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I > . >
TLC
FOR IDENTIFICATION
OF AMINO
COMPOUNDS
211
4--jJlti---
3
212
AJMAL,
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AND
ANWAR
FIG. 2. Sorption of amines on cellulose, alumina, and silica gel layers with the cyclohexanebenzene (l:l, v/v) solvent system. 1, m-PDA; 2, p-PDA; 3, p-NAL; 4, m-NAL; 5, I-NPA; 6, m-CAL; 7, AL; 8, DPA; 9, CZ; 10, p-CAL; 11, p-TD; 12, NJ,-DMAL.
cyclohexane-benzene (1: 1) solvent system. The lower RF values on silica gel layers may be attributed to the interaction between the acid surface group of the silica gel and the aromatic amines. This interaction may lead to the existence of
It is well known that hydrogen bonding plays a crucial role in binding the carcinogen to nucleic acid analogues of DNA (15). It is interesting to note from Fig. 2 that amines containing two aryl groups, i.e., DPA or CZ (RF = 0.95), can be selectively separated from amines containing one aryl group like aniline, substituted aniline, and aniline derivatives (RF = 0.0-0.35) on silica gel layers. The higher R, values for CZ and DPA may be attributed to their lower basicity. It is clear from Fig. 2 that most of the amines showed different selectivities toward the three adsorbents described above. Therefore, the retention behavior of amines on mixed adsorbents containing silica gel and alumina or cellulose is amenable to study. The R, values of amines in cyclohexane-benzene (1: 1) are illustrated in Fig. 3 as a function of the percentage concentration of silica gel in the mixture containing silica gel and alumina or cellulose as sorbent phase. It is evident from Fig. 3 that there are several possible selective separations of amines on mixed sorbent phases, for example, p-CAL from o-CAL on silica gel-alumina and silica gel-cellulose containing 33 and 50% silica gel, respectively. Similarly, silica gel-alumina containing 33% silica gel may be used to separate DPA from p-TD or o-NAL. Silica gel-cellulose containing 66% silica gel was found to be a good adsorbent for selective separation of DPA.
TLC FOR IDENTIFICATION
33 Concentrot~on
SilCO
of
gel
(*A)
50
66
33
of
concentration
S~l~CO gel
OF AMINO
(‘/.I
50
33
66
Concentrot~on SIIICO
213
COMPOUNDS
gel
of (%I
50
66
Concentrotlon Sil~CO
of
gelf%)
FIG. 3. RFvalues of amines as a function of the silica gel concentration in cyclohexane-benzene (1: 1, v/v). 0, Silica gel-alumina adsorbent; 0 silica gel-cellulose adsorbent.
Separations on TLC are sometimes hampered due to the appearance of tailed spots caused by high loading of sample. In order to determine the maximum amount of an amine which can be separated without tailing on silica gel-cellulose (2:l) with cyclohexane-benzene (I:l), we calculated the R, and RT values for variable concentrations of amines. Figure 4 indicates that most of the amines produce compact spots (RL - R, < 0.3) in the l-IO% concentration range (loading amount l-10 mg) of sample. p-NAL at 10% concentration and aniline at all concentrations (l-10%) gave tailed spots. Table 1 indicates that the proposed method for the identification of amines on TLC plates is reasonably sensitive. Aniline down to 0.05 u,g can be detected on TLC plates. The qualitative separations achieved on mixed adsorbent layers containing silica gel and alumina or cellulose with various solvent systems are summarized in Table 2. In addition to these separations, AL from m- or p-PDA, p-CAL, DPA from m- or p-PDA, p-NAL from NPA, m-PDA or p-PDA, p-CAL, and o-CAL from m- or p-PDA were achieved on plain silica gel layers in different solvent systems. The results for the separation of DPA from m-PDA or p-PDA at milligram levels are tabulated in Table 3: 500 t.r,gof DPA can be well separated from 10
214
AJMAL,
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AND
ANWAR
Loodhg
,O’,. 5% 25% Loodmg amount
FIG.
(l:l,
3% lmg)
10% 5% 2.5’1. Loading amount
IV. (mg)
amount
10% 5% 2.5% I% Loodmg amount (mgl
4. Effect of loading amount of amines on the R, values. Mobile phase, cyclohexane-benzene v/v); stationary phase, silica gel-cellulose (2:l, w/w). 0, R, values; 0, R, values.
mg of p-PDA concentration or m-PDA is separation of
and vice versa. The mutual separation range 500 pg-5 mg is possible. However, loaded, the spots are barely separated. o-NAL from p-NAL is not hampered
of DPA and m-PDA in the when 10 mg of either DPA Table 4 indicates that the by the presence of certain
TABLE 1 Limit of Detection of Amines on Silica Gel-Cellulose (2: 1) Layers with the Cyclohexane-Benzene (1: 1) Solvent System No.
Amines
Limit of detection w
1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13.
Aniline Diphenylamine p-Nitroaniline I-Naphthylamine o-Chloroaniline o-Nitroaniline m-Phenylenediamine p-Phenylenediamine p-Chloroaniline p-Toludine Carbazole m-Nitroaniline m-Chloroaniline
5.0 x 10-a 5.0 x 10-t 5.0 x 10-a 5.0 5.0 5.0 x 10-t 5.0 x 10-t 5.0 x 10-t 5.0 x 10-t 5.0 x 10-t 1.0 x 10-l 5.0 x 10-l 5.00
(mg I
TLC
Separation Stationary
FOR
of Amines
on Mixed
phase
Mobile
Silica gel G-AI,O, 2:1 2:1
1. 2.
IDENTIFICATION
OF
TABLE Adsorbent
AMINO
2 Layers
phase
with
Various
DPA (10.0 - 9.$po-NAL (7.0 - 5.0)
Benzene
o-CAL
(8.5 - 6.5)
4.
1:l
Benzene
o-CAL
(8.5 - 6.5)
5.
1:l
Benzene
o-NAL
(7.0 - 5.0)
6.
1:2
Benzene
DPA (9.0 - 8.0)
7.
I:2
Benzene
o-CAL
8.
2:1
Cyclohexone-benzene
(1: 1)
DPA (8.0 - 6.0)
9.
2:1
Cyclohexane-benzene
(1: 1)
p-NPA
(4.5 - 3.0)
10.
I:2
Cyclohexane-benzene
(1:l)
o-CAL
(7.5 - 5.0)
11.
I:2
Cyclohexane-benzene
(1:l)
DPA (8.0 ~ 7.0)
12.
1:l
Cyclohexane-benzene
(1: 1)
o-CAL
(2: 1) ( 1: 1) ( 1:2) ( 1: 1) (2: 1)
p-NAL (3.5 DPA (6.5 p-NAL (1.5 p-NAL (1.5 p-NAL (0.0 p-NAL (1.5 DPA (3.0 -
Separation
of DPA
Benzene Benzene Cyclohexane-benzene Cyclohexane-benzene Cyclohexene-benzene Cyclohexane-benzene Cyclohexane-benzene
TABLE m- and p-PDA at Milligram with the Cyclohexane-Benzene
from
Amount No.
Separation DPA*-p-PDA DPA*-p-PDA DPA*-p-PDA DPA*-p-PDA DPA*-m-PDA DPA*-m-PDA DPA*-m-PDA p-PDA-DPA p-PDA*-DPA p-PDA*-DPA m-PDA*-DPA m-PDA*-DPA m-PDA*-DPA
* Refers
to the amount
of corresponding
(9.0 ~ 7.5)
(7.0 - 5.0)
Levels
or m-PDA (0.5 - 0.0) -m-PDA (1.0 - 0.0) -p-PDA (0.5 - 0.0) -in-PDA (0.5 - 0.0) -p-PDA (1.0 - 0.0) -m-PDA (0.3 - 0.0) -p-PDA (1.0 - 0.0) -m-PDA (1.0 - 0.0) -p-PDA (1.0 - 0.0) -m-PDA (0.0 0.0) -p-PDA (0.0 - 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 ~ 0.0) -p-NAL (0.5 - 0.0) -m-PDA (0.5 - 0.0) -p-PDA (0.5 - 0.0) -p-NAL (0.10 - 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 - 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 - 0.0) -m-PDA (0.4 - 0.0) -p-PDA (0.3 - 0.0) -p-NAL (0.0 ~ 0.0) -m-PDA (0.0 - 0.0) -p-PDA (0.0 - 0.0) -o-NAL (7.0 - 6.0) -cz (10.0 - 9.5) -o-NAL (4.5 - 3.0) -o-NAL (5.5 - 3.0) -o-NAL (4.0 - 3.0) -o-NAL (5.0 - 3.0) -cZ (9.0 - 8.0)
on Silica Gel-Cellulose (I : 1) System
(2: 1) Layers
loaded
0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5*
10 (RL 1 5 10 12 1 5 10 1 5 10 1 5 IO
-
- 2.0) 3.5) ~ 1.0) ~ 0.0) - 0.0) - 0.5) 2.0)
- &I
3
(mg)
R,
Phases 10 CR,
Benzene Benzene
2:l
Silica-cellulose 1:l 1:2 1:l 1:2 1:2 2:1 1:2
Mobile
Separations
3.
1. 2. 3. 4. 5. 6. I.
215
COMPOUNDS
R,- values
for DPA.
- RJ
(IO - 8.5)*, (2.5 - 0) (9.8 - 8.6)*, (4.0 - 0) (10 - 7.5)*, (5.5 - 0) (IO - 7.8)*, (6.0 - 0) (IO - 9)*, (3.0 - 0) (IO - 9)*, (4.0 - 0) (IO - 9)*. (9 - 0) (1.5 - O)*, (9.8 - 9.0) (6 - O)*, (10 - 6.5) (2.5 - O)*, (9.6 - 8.6) (3.0 - o)*, (IO - 9) (5.0 - o)*, (IO - 9) (7.0 - oj*, (10 - 7)
216
AJMAL,
MOHAMMAD, TABLE
AND
ANWAR
4
Separation of o-NAL from p-NAL in the Presence of Metal Salts on Silica Gel-Cellulose Layers in the Cyclohexane-Benzene (1: 1) System No.
Metal salt
1.
Sodium chloride
2.
Copper sulfate
3.
Ammonium chloride
4.
Nickle chloride
5.
Cobalt sulfate
6.
Chrominum chloride
I.
Manganese chloride
8.
Vanadium sulfate
Amount added (mg) 10.0 30.0 45.0 10.0 30.0 50.0 10.0 30.0 50.0 10.0 30.0 50.0 10.0 30.0 10.0 30.0 40.0 10.0 30.0 50.0 10.0 30.0 40.0
a RL - RT values for the separation of o-NAL and p-NAL o-NAL (3.8 - 2.8) andp-NAL (1.5 - 0.5).
p-NAL” 10 (RL - JW (0.5 (0.8 (0.8 (0.5 (0.8 (0.5 (0.5 (0.8 (0.5 (0.5 (0.8 (0.6 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5 (0.5
-
0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0) 0)
(2: 1)
o-NAL” 10 (RL - &) (4.0 (4.0 (3.1 (3.5 (4.0 (2.8 (3.0 (4.2 (2.6 (3.0 (4.8 (3.0 (3.0 (3.0 (3.0 (3.3 (3.0 (4.0 (3.5 (2.5 (2.6 (2.6 (2.8
-
3) 3.0) 1.6) 2.5) 3.0) 2.0) 2.0 3.2) 2.0) 2.0) 3.8) 2.0) 2.2) 2.2) 2.0) 2.2) 2.0) 3.0) 2.5) 1.4) 2.0) 2.0) 1.8)
in the absence of salt are as follows:
metal salts up to 45-50 mg. However, the tolerance of cobalt sulfate is only up to 30 mg. The amines cannot be detected when the loading of metal salts exceeds the limiting value noted in Table 4. An increase in resolution was observed in the presence of metal salts compared to the separation of o-NAL from p-NAL in the absence of metal salts from the synthetic mixture. ACKNOWLEDGMENTS The authors thank Professor K. T. Nasim for providing research facilities. S.A. thanks the U.G.C. for financial assistance.
REFERENCES 1.
2. 3. 4. 5. 6.
Nabi, S. A.; Moharnmad, A.; Qureshi, P. M. Talanta, 1979, 26, 1179. Qureshi, M.; Nabi, S. A.; Khan, I. A.; Qureshi, P. M. Talanta, 1982, 29, 757. Papadoyannics, I. N. Microchem. J., 1985, 32, 220. Cserhati, T.; Bordes, B.; Szogyi, M. J. Chromatogr. Sci., 1986, 24, 302. Khulbe, K. C.; Mann, R. S. Fresenius Z. Anal. Chem., 1988, 330, 642. Gennaro, M. C.; Marengo, E. Chromatographia, 1988, 25, 603.
TLC FOR IDENTIFICATION
OF AMINO COMPOUNDS
7. Iskander, M. L.; Medien, H. A. A.; Nashad, S. Microchem. J., 1987, 36, 368. 8. Singh, D. K.; Darbari, A. Chromatographia, 1987, 23, 93. 9. Dhillan, R. S.; Singh, J.; Gautam, V. K.; Chhabra B. R. J. Chromarogr., 1988, 435, 256. 10. Kuhn, A. 0.; Lederer, M. J. Chromatogr., 1988, 440, 165. 11. Geerdink, R. B. J. Chromatogr., 1988, 44.5, 237. 12. Phars, D. Y.; Laiden, P. C.; Siggia, S. Chromatogr. Sci., 1985, 23(9), 391-396. 13. Hovea, N.; Elena, M.; Teodar, H. Rev. Chim., 1985, 36, 446. 14. Dimov, N.; Agapona, N.; Nauchnoizseled, Tr. Khim Farm. Inst., 1986, 16, 113. IS. Hathway, D. E.; Kolar, G. F. Chem. Sot. Rev., 1980, 241.
217