477
Anal. Chem. 1981. 53,477-480
spiked into an oil sample which was then subjected to the entire esterification procedures, essentially all the alcohol was recovered.
Table I. Analysis of Oleic Acid in Dewaxed Petroleum Oil" amt of re1 int oleic acid re1 amt of areab of amt of spiked, mg oleic acid m/z = 296 oil, mg 167.7 161.7
13.5 12.1 7.6
1.o
LITERATURE CITED Drozd, J. J . Chromatogr. 1075, 113, 303-305. Sheppard, A. J.; Iverson, J. L. J . Chromtcgr. Scl. 1075, 73, 448-450. Krupcik, J.; Hrivrak, J.; Janak. J. J . Chromtcgr. S d . 1076, 14, 4-7. Luddy, F. E.; Barford, R. A.; Herb, S. F.; Megktem, P. J . Am. 011 Chem. Soc. 1068, 45, 549-552. Chrlstopherson, S. W.; Glass, R. L. J . Dslry Sc1. 1060, 5 2 , 1289- 1290. Blank, M. L.; Verdino, 8.; Drtvett, 0. S. J. Am. 0llChern. SOC.1065, 42, 87-90. Peisker, K. V. J. Am. 011 Chem. Soc. 1064, 41, 87-92. Matcalf, L. D.; Schmitz, A. A.; Peke, J. R. Anal. Chem. 1066, 3 8 , 5 14-51 7. Lloyd, J. 0 . F.; Roberts, B. R. 0. J . ChrOmatOgr. 1073, 77, 228-231. (;rWieY, R. H. J . Chromstwr. 1074, 88, 229-231. West, J. C. Anal. Chem. 1075, 47, 1708-1711.
1.o
0.9
0.9
0.6
0.6
1 pL of sample injected each time at a split ratio of 75:l. A 30-m glass SP-1000 WCOT column was used and the column temperature was kept at 210 "C isothermally. An average of five runs.
in a used petroleum oil sample. Clearly a series of n-carboxylic acids from C7 to C20 are the predominant components present in this used oil sample. We have also found that the esterification procedures described above are very selective. For example, other oxygenated species such as fatty alcohols and aldehydes in the same used oil sample were not altered. When octadecenol was
RECEIVED for review May 28,1980. Accepted November 6, 1980. Presented a t the 28th Annual Conference of Mass Spectrometry and Allied Topics, New York, May 2630,1980.
Identification of Macrocyclic Polyamines and Macrocyclic Dioxopolyamines by Thin-Layer Chromatography, Gas Chromatography, and Electrophoresis Takashl Yatsunaml, Atsuko Sakonaka, and Elichi Kimura' Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Kasumi, Hiroshima, 734, Japan
Simple and rellable ldentlflcatlon methods for Synthetic macrocyclic polyamlnes and macrocycllc dloxopolyamlnes of chemical slgnlflcance have been establlshed by using thlnlayer chromatography, gas chromatography, and electrophoresis. The following TLC systems were effectlve: (a) butanol-acetic acld-water (4:1:5) on alumlna and (b) chloroform-methanol-28 % ammonia (2:2:1) on silica gel. Macrocyclic polyamlnes were also analyzed by GC on a Aplezon L or a Thermon 1000 KOH column or by electrophoresls uslng buffers contalnlng dl- or trlcarboxylates at pH -6. Anomalous electrophoretlc behavior of some macrocycllc polyamines mlgratlng to anode dlrectlon In cttrate buffer solutlon at pH 6 was dlscovered.
+
The chemistry of synthetic macrocyclic polyamines and macrocyclic dioxopolyamines L1-L19has been drawing much interest (1-7). These macrocycles form much more stable and selective complexes with various transition-metal ions than do open chain analogues having the same donor arrangement. The macrocyclic complexes present great potential as models of metalloenzymes (8-11), sequestering agents for specific metal ions ( 4 , 6 ) , synthetic reagents (12), or biomedical applications (13). However, analytical methods for detection and identification of these macrocycles have not been well established, which very often hamper their further application. We report here simple and convenient methods for qualitative and quantitative analyses of various macrocyclic polyamines and macrocyclic dioxopolyamines using thin-layer (TLC) and gas chromatography (GC) and electrophoresis. 0003-2700/81/0353-0477$01.00/0
The inherent difficulties in analysis of these compounds are partly ascribed to their highly polar and basic characters caused by more than three amine and amide groups. The success of the present work relies upon the careful selection of solvents (butanol-acetic acid-water (4:1:5) or chloroformmethanol-28% ammonia (2:2:1)) in TLC, liquid phases (Apiezon L or Thermon lo00 + KOH) in GC, or electrolyte solutions (di- or tricarboxylates, pH -6) in electrophoresis, whereby the macrocycles having only slightly deviating structures are well separable from each other. The present methods can also distinguish macrocycles from closely related linear polyamines and the group of macrocyclic polyamines from those of macrocyclic dioxopolyamines, respectively. This is quite significant, since the former are very often synthesized from the latter as starting materials.
EXPERIMENTAL SECTION Materials. The macrocyclic triamines L1 (as L1.3HCl) and L2 (as L2.3HBr) were prepared by the method of Koyama and Yoshino (14). The macrocyclic tetramines L,-L8 (as 4HC1 salts), and hexamine L13 (as L1&HCI) were synthesized by the method of Martin et al. (15). The macrocyclic triamine L3 (as L3.3HBr), tetramine (as b.4HBr) and Ll0 (asLlo-4HBr),and pentamine Lll (as L11.5HCI)and L12 (as L12.5HBr)were prepared according to the methods described before (13, 16). All the macrocyclic dioxotetra- and dioxopentamines were obtained according to the slightly modified procedures by Tabushi et al. (17)and Kato (18). Their structures and purities were checked by elemental analysis, 'H NMR, and melting point. Free bases of L1-L13 were obtained as follows. The amine salts were dissolved in water, basified with 2 N NaOH, and extracted with chloroform four times. Combined chloroform layers were dried with sodium sulfate and evaporated to dryness. All the other solvents and chemicals were of analytical 0 1981 American Chemical Society
478
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
L1
L2
9 - a n e - N3
1 0 - a n e - N3
L3
13-ane- N~
L8
L7
15-ane- N4
L11
15- a n e - N5
L16 dioxo-14-ane-Nq
16-ane- N4
L13
L12
18 - a n e
1 6 - a n e - N5
L1 7
dioxo-15-ane
- N4
- Ne
L4 1 2 - a n e - N4
L5 13-a n e - ~4
L9
L10 17- a n e N4
i s 0 -16
- ane- N 4
L14
d ioxo -1 2 -ane - N4
L18 d !oxo -1 5- a n e "5
-
- L15
d ioxo 13- ane- N4
L19 d ioxo-16- ane- N5
Flgure 1. Synthetic macrocyclic polyamines and dioxopolyamines.
-
Table I. Rf Values of Macrocyclic Polyamines, Macrocyclic Dioxopolyamines, and Related Linear Polyamines Using System A (Alumina, Butanol-Acetic Acid-Water (4:1:5)) and B (Silica Gel, Chloroform-Methanol-28% Ammonia (2:2:1)) Rf
-
polyamines macrocyclic polyamines g-ane-N, (L,) 10-ane-N, (L,) 13-ane-N, (L,) 12-ane-N, (L,) 13-ane-N, (L,) 14-ane-N, (L,) 15-ane-N, (L,) 16-ane-N, (L,) iso-16-ane-N, (L,) 17-ane-N, (Ll,,) 15-me-N, (L,,) 16-ane-N, (LI2) 18-ane-N, (Ll,)
A
0.46 0.49 0.54 0.46 0.54 0.62 0.43 0.31 0.38 0.31 0.46 0.46 0.39
B 0.01 0.04 0.18 0.04 0.01 0.01 0.04 0.02 0.01 0.01
polyamines macrocyclic dioxopolyamines dioxo-12-ane-N, (L,,) dioxo-l3-ane-N, (L,,) dioxo-14-ane-N, (L16) dioxo-15-ane-N, (L,,) dioxo-15-ane-N, (LIB) dioxo-l6-ane-N, (L,9) linear polyamines 2,2-tria 3,3-trib 2,2,2-tetc 2,3,2-tetd 3,3,3-tete 2,2,2,2-pent f
a 1,4,7-Triazaheptane. 1,5,9-Triazanonane. 1,4,7,10-Tetraazadecane. Tetraazatridecane. f 1,4,7,10,13-Pentaazatridecane.
reagent grade and were used without further purification. TLC Procedures. TLC was performed on silica gel 60Fm precoated plates of 0.25 mm thickness (Merk) or alumina plates of 0.2 mm thickness prepared in our laboratory by using aluminum oxide G, Type E (Merk). One microliter of ethanolic or aqueous solutions containing 10 pg/pL of the free bases of amine salts was applied to the plates. Separations were carried out in a conventional chromatographictank containing Toyo No. 2 filter paper on a surface. The spots were visualized with iodine vapor. R, values obtained were shown in Table I. GC Procedures. A Shimazu GC-4CM gas chromatographwith dual-flame ionization detectors was employed for the analysis. Two types of silaniied glass columns were used. One was a column
A
B
0.38 0.54 0.42 0.51 0.31 0.46
0.62 0.58 0.55 0.62 0.60 0.40
0.30 0.23 0.27 0.21 0.16 0.21
0.05 0.05
1,4,8,11-Tetraazaundecane.
0.04 e
1,5,9,13-
of 1 m X 3 mm packed with 5% Apiezon L on Chromosorv W (AW-DMCS, 80-100 mesh) and another was that of 1.5 m X 3 mm packed with 10% Thermon lo00 + 3% KOH on Chromosorv W (AW-DMCS,80-100 mesh). Both columns were set at 240 OC for 12 h before use. The flow rate was 40 mL/min throughout the work. Column temperatures were indicated in the text. One microliter of ethanolic solutions containing 5 pg/pL of the free bases was injected. The resulting retention times are listed in Table 11. Electrophoresis. Electrophoresis was performed by using a Gelman semimicroelectrophoresis chamber and a Atto VC-stabilizer SJ-1051. Gelman Sepraphore 111 (6 X 22 cm) was used as supporting medium for each run. The electrophoresis strips
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
479
Table 11. Retention Times (min) of Macrocyclic Polyamines, Macrocyclic Dioxopolyamines, and Related Linear Polyamines liquid phases (temp, “C) liquid phase (temp, “C) Thermon Thermon 1000 1000 Apiezon La KOH Apiezon La KOH
;
macrocyclic triamines 9-ane-N, (L,) 10-ane-N, (L,) 13-ane-N, (L,) linear triamines 2,2-tric 3,3-tri macrocyclic tetraamines 12-ane-N, (L,) 13-ane-N, (L,) 14-ane-N, (L,) 15-ane-N, (L,) 16-ane-N, (L,) iso-16-ane-N4 (L,) 17-ane-N, (Llo) a
(150) 2.2 3.5 9.9 (150)
f180) 3.0 4.3 7.2 (180)
1.4 (200) 2.4 2.9 3.8 4.9 6.9 7.5 11.3
3.2 (220) 4.7 4.8 5.1 6.7
1.6
linear tetraamines 2,2,2-tet 2,3,2-tet 3,2,3-tetd 3,3,3-tetc macrocyclic pentaamines 15-ane-N, (L,, ) 16-ane-N, (L12) linear pentaamine 2,2,2,2-pentc macrocyclic hexaamine 18-ane-N, (L,,)
8.3
8.7 12.6
5% on Chromosorb W (80-100mesh), 1.0 m long, 3 mm i.d., flow rate 40 ml/min. See footnote in Table I.
(80-100 mesh), 1.5 m long, 3 mm i.d., flow rate 40 ml/min.
were connected to the electrode vessels by bridges which consist of two layers of Toyo No. 50 filter paper. After equilibration for 15 min, 1pL of aqueous solutions containing 10 g / p L of the free bases or amine salts was applied on the strips for elution by the specified potential. The polyamine spots were detected by staining the strips with iodine vapor. Electrolyte solutions used were as follows: (a) formic acid-acetic acid, pH 2; formic acid (85%)7.1 mL, acetic acid (99.5%) 21.9 mL, and water to make a 500-mL solution, adjusted to pH 2 with sodium hydroxide; (b) phosphate-borate, pH 8.4; potassium biphosphate 1.46 g, borax 3.24 g, and water to 500 mL solution; (c) acetic acid-NaOAc, pH 2; 0.1 M acetic acid 27 mL and 0.1 M sodium acetate 491 mL; (d) lactic acid-NaOH, pH 4.9; 0.1 M lactic acid 50 mL, 0.1 M sodium lactate 435 mL, and water to 500 mL solution; (e) succinic acidNaOH, pH 5.8; 0.1 M sodium hydrogen succinate 167 mL, 0.1 N sodium hydroxide 119 mL, and water to 500 mL solution; (0 phthalic acid-NaOH, pH 6; 0.1 M potassium hydrogen phthalate 167 mL, 0.1 N sodium hydroxide 132 mL, and water to 500 mL solution; (g) tartaric acid-NaOH, pH 5.3; 0.1 M tartaric acid 167 mL, 0.1 N sodium hydroxide 317 mL, and water to 500 mL solution; (h) citric acid-NaOH, pH 6; 0.1 M citric acid 83 mL, 0.1 N sodium hydroxide 233 mL, and water to 500 mL solution. Tables I11 and IV summarize the results of the electrophoretic separations of the macrocycles.
RESULTS AND DISCUSSION TLC Separation. A number of developing solvents including aqueous and nonaqueous systems were tested for the macrocyclic polyamines LI-L13 and macrocyclic dioxopolyamines L14-LI9 on silica gel or alumina plates. The following combinations were found to be most effective: (a) butanolacetic acid-water (4:1:5) on alumina, (b) chloroform-methanol-28% ammonia (2:2:1) on silica gel. Table I shows the R, values of the macrocycles L1-L19 and of the relevant linear polyamines. System a gave excellent separations of the majority of macrocyclic polyamines, macrocyclic dioxopolyamines, and linear polyamines. The spots are visualized on exposure to iodine vapor and have appropriate R, values for identification work. System b can well separate the group of macrocyclic dioxopolyamines from those of macrocyclic polyamines and linear polyamines, which remain almost a t the starting points. A combination of the two systems has served as a very useful tool for synthesis of macrocycles in our laboratory. GC Procedures. The macrocyclic polyamines L1-L13 can be analyzed by GC without prior derivatization of the original structures. Excellent separation of the macrocyclic polyamines from each other and from linear polyamines was achieved with a nonpolar Apiezon L column. Macrocyclic dioxopolyamines
10% + 3% on Chromosorb W 1,5,8,12-Tetraazadodecane.
Table 111. Mobilities of Macrocyclic Polyamines, Macrocyclic Dioxopolyamines, and Related Linear Polyamines Relative to 14-ane-N, at Various pHs formic citric acidacidphosphateacetic acid. NaOH. borate. compound ~ H 8 . 4 ~ pH 2a ’ p H 6 6 1 0-ane-N, (L,) 13-ane-N, (L,) 12-ane-N, (L,) 13-ane-N, (L,) 14-ane-N, (L,) 15-ane-N, (L,) iso-16-ane-N4 (L9)
17-ane-N, (Lid
dioxo-12-ane-N4 @I,)
dioxo-14-ane-N,
1.00
1.00
1.04 1.09
0.54 -0.19d
0.59 0.93 1.01 0.99 1.00 1.00 0.89
1.02
-0.4gd
0.82
0.96
-0.86d
0.67
0.97
0.39
0.55
0.83
0.32
0.76
0.36
0.76
0.86
0.30
0.78
1.31 1.24 1.34
0.72 0.04 0.54
1.10
1.05 1.01
(L16)
dioxo-15-ane-N. (L17)
dioxo-16-ane-N5 (LI& 3,4-trie 2,3,2-tetf 3,4,3-tetR
0.76 0.91 1.16 1.05
0.89 0.86 0.80 a 200 V, 30 min. 300 V, 10 min. 300 V, 30 min. A sign of minus (-) before a value of mobility indicates that movement is toward anode. e 1,5,10-”riazadecane, f See footnote in Table I. 1,5,10,14-Tetraazatetradecane. L14-L19 failed to be detected, probably due to thermal decomposition. The retention times are listed in Table 11. Apiezon L achieved a complete separation of macrocyclic polyamines Li-Li3 and the order of elution parallels those for their molecular weight. Thermon loo0 KOH also gave fairly good separation only except for L4, L5, and Le Quantitative analysis also was possible, since the peaks recorded on both the columns are sharp and well-defined. Other columns such as OV-17 and SE-30 gave poor resolution of the amines or resulted in tailing of the peaks. Electrophoresis. We tested the electrophoretic separabilities of the macrocycles on cellulose acetate membranes at
+
480
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
Table IV. Influence of Carboxylic Acids in Electrolyte Solutions on the Mobilities of the Macrocyclic Polyamines Relative to 14-ane-N4
electrolYtesolution acetic acidNaOAc (pH 6 ) a lactic acidNaOH (pH 4.9)b succinic acidNaOH (pH 5.8)a phthalic acidNaOH (pH 6)c tartaric acidNaOH (pH 5.3)c citric acidNaOH (pH 6 ) d a
300 V, 5 min.
macrocyclic polyamines 10-ane- 13-ane- 12-ane- 13-aiie- 14-ane- 15-ane- iso-16-ane- 17-aneN, (L,) N3 (L3) N4 (L4) N4 (Ls) N, (L,) N4 (L,) N4 (L,) N4 (Llo) 1.10 1.08 1.18 1.11 1.00 0.93 1.02 1.05
SaneN,
(LIS)
1.09
1.11
1.09
1.16
1.09
1.00
0.98
1.00
0.93
0.86
1.12
1.02
1.07
1.05
1.00
0.91
0.77
0.62
0.51
1.25
1.33
1.21
1.00
0.83
0.47
0.29
0.25
1.21
1.00
0.82
0.68
0.55
0.03
1.05
1.00
0.54
-O.lge
--0.4ge
-0.86e
1.30
1.23
0.76
0.91
300 V, 6 min.
1.16
300 V, 8 min.
300 V, 10 min.
Figure 2. Proposed model for 1:l complex of L,3 with citrate.
several different pHs using various electrolyte solutions, since the macrocycles possessing a wide range of positive charges should show varied mobilities. Table I11 summarizes the results of three representative separations of the macrocycles and the related linear polyamines, The best separation of the macrocyclic polyamines L2-L13 was achieved with citrate buffer solution at pH 6, wherein all of the spots were welldefined and widely scattered on the stained strips. On the other hand, the macrocyclic dioxopolyamines could not be resolved with any electrolyte solution tested. More noteworthy with the citrate system is the occurrence of anomalous retardation of the mobilities at larger sizes of the macrocyclic polyamine rings. This becomes most obvious for 16- to 18-membered macrocycles (Lg,Llo, and L13), which were found to migrate to the anode, the opposite electrode direction for the anticipated cations (by protonation). Some retardation was also reported in the electrophoresis of amino acids (19), which was ascribed to complex formation with metal ions in electrolyte solutions. However, such peculiar migration of the cations toward the opposite electrode direction has not been found in the literature, to the best of our knowledge. On the basis of the assumption that the carboxylate anion used as electrolyte might be responsible for this anomaly, we have examined the effects of mono- and dicarboxylates on the mobilities of the macrocyclic cations at pH -6. The results are presented in Table IV, which shows greater retardation with larger number of carboxyls on the carboxylic acids (order of retardation: acetic C lactic C succinic C phthalic = tartaric < citric acid). As an extreme case, citric acid (having three carboxyls) caused the reversed migrations for Lg, Llo, and L13. A tentative explanation for the present finding with polycarboxylate buffers is complex formation with di- or triprotonated macrocycles near pH 6 through hydrogen bonding, which may effectively shield the positive charge of the macrocycles. At pH 6 the macrocycles strongly hold two or three protons depending on the ring size within the rings (20), and the carboxylic acids are dissociated as di- or trianions. The
e
See footnote in Table 111.
pKa values, for instance, are 11.2, 10.0, C2,
