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Chapter 16 Alkaloids
395
Chapter 16 ALKALOIDS
2. DEYL A l k a l o i d s a r e n i t r o g e n o u s bases o f p l a n t o r i g i n and i n c l u d e a l m o s t a l l o f t h e known n i t r o g e n - c o n t a i n i n g r i n g systems. A g i v e n p l a n t s p e c i e s v e r y seldom c o n t a i n s a s i n g l e a l k a l o i d and u s u a l l y m i x t u r e s o f a l k a l o i d s w i t h s i m i l a r b i o l o g i c a l prope r t i e s a r e found t o g e t h e r . Commercial p r e p a r a t i o n s a r e u s u a l l y crude m i x t u r e s c0ntainin.g v a r y i n g amounts o f a l k a l o i d s and zone e l e c t r o p h o r e s i s o n paper has been w i d e l y a p p l i e d t o t h i s c a t e g o r y o f compounds. A1 k a l o i d s g e n e r a l l y m i g r a t e as c a t i o n s o v e r t h e pH range 1.8-12.5. TABLE 16.1 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS AT DIFFERENT pH VALUES (REF. 12)a A1 k a l o i d
A pomorph ine Atropine Caffeine Cocaine Ergotamine Heroin Lobe1 i n e Morphine Nicotine Papaveri ne Quinine Scopolamine Sparteine Strychnine Theobromine Tubocurarine a
M i g r a t i o n (cm) a t pH
1.8
2.1
-
8.4 12.4 - 3.9 - 14.0 - 7.5 - 13.7 - 12.5 - 13.8 - 24.3 - 11.4 - 17.9 - 11.9 - 20.1 - 11.8 - 4.4 - 15.0
10.0 14.1 - 5.9 14.6 6.8 - 13.6 - 12.6 - 14.0 27.8 - 12.6 - 20.2 - 14.0 - 22.1 - 13.4 - 4.3 - 14.9
-
-
-
-
-
4.2
-
7.2 14.0 - 5.1 - 16.3 - 7.2 - 15.2 - 14.2 - 15.3 - 18.3 - 12.1 - 14.9 - 13.0 - 18.8 - 12.6 - 5.2 15.3
-
.
6.4
-
5.2 14.3 - 6.4 14.5 - 4.5 - 12.7 - 12.7 - 13.1 17.2 - 7.6 - 11.5 - 14.6 - 15.8 - 12.1 - 6.7 - 12.7
-
-
-
8.6
-
4.2 15.2 8.0 - 14.7 - 6.2 12.0 - 13.2 - 12.0 - 13.0 - 5.0 10.2 - 11.6 - 16.8 - 10.5 - 8.4 11.8
-
-
-
-
-
11.2
-
2.0 13.7 - 8.5 9.9 - 4.4 - 7.0 - 4.4 - 7.2 - 7.5 - 5.1 - 7.2 8.7 - 16.7 - 8.0 - 6.1 - 8.3
-
-
-
12.5 1.0 - 6.0 - 3.5 - 10.1 - 6.3 - 7.6 - 7.4 - 7.9 - 3.5 - 3.0 - 3.0 - 4.0 - 9.5 - 3.0 - 6.5 - 9.0
B r i t t o n - R o b i n s o n phosphate-acetate-borate (0.04 m o l / l o f each) b u f f e r was used between pH 1.8 and 11.2 and a s o l u t i o n o f g l y c i n e a t pH 12.5; S c h l e i c h e r and S c h i i l l 2043 b paper ( 3 x 60 cm); 500 V ( 8 V/cm) f o r 3 h; movement i s towards t h e cathode except f o r apomorphine a t pH 12.5
396
TABLE 16.2 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS AT DIFFERENT pH VALUES (REF. 13)a ALKALOID
M i g r a t i o n (cm) i n b u f f e r b o f pH 2.3
Apomo r ph in e Arecol i n e Aspidospermine Atropine Berberine Bicuculline Bol d i n e Brucine Bul bocapnine Caffeine Cal ebassine Chel i d o n i n e C i n c h o n i d i ne Cinchonine Cocaine Col c h i c i n e Coniine C o r l umine Co r y d ine Curarine Cotarnine Dicentrine Dimet h y 1 b e r b e r ine Dinethyl tubocurarine Emetine Ephedrine Ergomet r ine Eserine F1 u o r o c u r i n e Gal eg ine Glaucine Harrnine Hero i n Homatropi ne Hydrast ine Hydrast in ine L-Hyoscyamine Jervine Lupan ine Lupinine Ma va c u r ine llescal i n e Mo r ph i ne Narceine N a r c o t i ne Nicotine Pa paver ine Pilocarpine P i per ine
- 4.2 - 8.8 - 5.24 - 6.3
- 1.5 - 5.1 - 2.2 - 4.5
-
- 3.7 - 0.3 - 6.5 - 4.95
10.4 10.2 6.1 0.2 8.9 4.95 4.6 10.6 8.4 1.5 6.6 6.8 7.5 8.0 3.5 6.15 7.7 9.1 2.8 @,.l 5.3 6.3 5.05 9.5 6.6 4.1 5.8 8.0 3.1 7.4 - 5.8 4.0 5.0 18.0 4.7 7.0 0
-
4.3
6.4
- 9.75 - 5.7 - 6.6 - 2.7 - 5.0 - 2.5 - 4.6 - 3.5 - 1.2 - 4.55 - 6.8 - 6.8 - 6.8 - 0.8 - 8.9 - 4.95 - 4.6 - 9.8 - 8.5 0 - 7.9 - 6.95 - 6.6 - 10.0 - 3.6 - 6.5 - 13.0 - 2.7 - 3.1 - 6.0 - 8.4 - 5.4 - 9.9 - 6.6 - 3.9 - 7.35 - 8.3 -
-
-
7.05 6.25 1.3 5.3 9.2 5.0 8.5
0
8.2
-. -13.5 8.7 10.7 - 2.0 6.2 3.0 6.9 3.3 2.35 8.0 5.8 9.3 8.0 - 11.0 2.1 13.0 5.9 7.1 11.0 12.5
-
-
-
0 - 10.4 - 9.9 - 8.5 - 10.0 - 3.8 - 10.5 - 8.0 - 11.8 - 3.8 2.6 - 9.5 - 10.4 - 6.1 - 13.2 - 10.3 - 5.3 - 10.6 - 12.1 - 2.4 - 10.2 - 10.0 - 2.6 - 5.4 - 12.3 - 3,o - 10.6 -
0.4
-
-
-
-
-
10.5 0 9.0 0 8.4 1.3 0 1.3 4.2 1.7 2.0
9.0
0 5.7 5.1 5.5 1.95 12.0 0 2.7 10.3 11.3 0 9.45 2.0 5.8 11.9 - 1.5 6.2 - 9.0 12.0 1.2 0 5.2 10.5 0 12.5 - 8.8 0 9.3 10.5 3.0 - 9.3 - 6.5 1.7 0 8.7
-
-
1.1 2.8 0 8.0 1.05 0 0 2.1 0.6 2.3 7.8 0 - 3.2 3.0 5.3 1.7 10.0 0 .- 1.4
-
-
-
-
-
-
-
--
-
10.6
- -1.3 - 5.0
- 10.0
4.0 0
-
-
10.8 0 7.8 8.7
10.8 - 0.3 0 2.85
0 - 11.3 6.9 0 8.7 10.4 2.4 8.2 2.85 - 1.65 0 - 4.5 0 1.95 0
-
0 1.5 0.9 0 3.8
-
-
-
0
-
-
-
-
-
-
-
-
-
-
-
11.4
-
-
-
-
-
1.3 0.3 5.0
0 0 0
1.5
-
2.1
-
0 6.4 4.5 0 5.3 4.9 0 2.2 1.5 0.9 or.2 6.8
0 0 0
1.5 0 4.5 1.8
r
1.8
- 1.3 - 1.9
-
1.2 1.3 1.5, 0 1.6 0 2.3 0
0.5
397 TABLE 16.2 (continued) ~~
.41k a l o i d
Protopine P r o t o v e r a t r i ne Quinine Sanquinarine Scopol ami ne Sempervirine Sol an ine Sparteine S t r y c hn ine Tetra hydropal ma t i n e Theobromine Theophyll i n e Tropacocaine Tropine Tubocurarine V e r a t r i ne Yohimbine
~
H i g r a t i o n (cm) i n b u f f e r b o f pH
2.3
4.3
6.4
8.2
- 4.6 - 3.5 - 10.2 - 1.5 - 6.7 - 0.8 - 2.65 - 11.5 - 5.6 - 4.5 - 0.45 - 0.25 - 6.7 - 7.5 - 7.0 - 4.0 - 4.5
- 5.0 - 3.5 - 6.8 - 1.38 - 6.65 - 0.7 - 2.9 - 10.0 - 5.85 - 4.2 - 0.8 - 1.0 - 8.6 - 6.6 - 6.7 - 3.7 - 4.7
- 7.7 - 5.85 - 6.5 - 4.8 - 7.6 - 4.6 - 1.1 0 - 10.4 - 6.0 - 0.5 0 - 4.0 - 4.0 - 10.8 - 10.1 - 8.5 - 5,2 - 4.1 0 - 2.1 - 1.8 - 2.1 0.9 - 10.2 - 10.6 - 14.5 - 5,85 - 8.4 - 5.8 - 6.2 - 3.8 - 6.8
10.5
11.4
- 3.9 - 2.5 0 - 3.3 -
0 0 0
-
0 0 9.9 0
0
1.3 0 0 4.7
--
0
- 1.8 1.15 - 8,8 0 - 4,7 .4 -- 11.0
0
2.0 5.5 2.0 0 0.7 0 0
aWhatman No. 1 paper; 8 V/cm f o r 3 h. bUniversal Britton-Robinson b u f f e r d i l u t e d 1:4. Movement i s towards t h e cathode i n most instances, as i n d i c a t e d by t h e minus signs.
TABLE 16.3 ELECTROPHORETIC MOBILITIES OF ALKALOIDS AT pH 5.0 AND 9.2 (REF. 8 ) A1 k a l o i d
Mobilitya i n b u f f e r o f DH 5.0b
Anabasi ne Anatabine N-Methylanabasine N-Methyl anatabine Metani c o t i n e Methylmetanicotine Myosmi ne
A1 k a l o i d
5.0b
9.2'
- 10.7 - 10.5 - 10.1 - 10.0 - 12.7 - 12.5 - 8.0 -
6.1 2.3 0.8 0.8 7.7 7.0 0.8
Mobilitya i n b u f f e r o f pH
Nicotine Nicotone N i co t y r i ne Nornicotine Norni c o t y r i ne Oxy nic o t ine
9.2'
-
12.1 - 3.4 - 3.2 - 12.2 - 1.5 - 3.9 -
;Mobility x lo5 cm2 V - l sec-' a t 10°C towards t h e cathode. Potassium hydrogen p h t h a l a t e b u f f e r (pH 5.0). Although t h k c o n c e n t r a t i o n o f b u f f e r i s n o t g i v e n i n r e f . 8, i t i s thought t h a t i t was 0.05 mol/l. '0.025 m o l / l borax s o l u t i o n (pH 9.2).
2.5 0.7 0
7.0 0.7 0.7
398
TABLE 16.4 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS I N 5 mol/l ACETIC ACID' A1 k a l o i d
Migrationa (cm)
Aconi t i ne Atropine Bruci ne Caffeine Cinchoni ne Coni i n e Ephedrine
- 3.2 - 4.5 - 4.0 - 0.5
-
-
-
8.0 10.5 5.2
A1 k a l o i d
Migrationa (cm)
Hyoscyami ne Lobe1 ine N i co t i ne Quinine Sol ani ne Strychnine Sparteine
-
-
4.6 4.6 9.9 7.4 3.4 4.8 7.6
aMigration towards the cathode; 5 mol/l a c e t i c acid; 750 V f o r 2 h.
Universal s e r i e s o f Britton-Robinson b u f f e r s can be used f o r zone electropho1 r e s i s on paper. By aFplying a high voltage i t i s possible t o separate, e.g., candicine, tembetarine, N-methyl isocorydine and n i t i d i n e 2 Good separations a r e
.
obtained a t pH 8.0 w i t h l a u r i f o l i n e , magnoflorine, berberine, palmatine and t h e benzophenanthridines. A t t h i s pH i t i s also p o s s i b l e t o d i f f e r e n t i a t e between cuscohygri ne, a t r o p i n e , hyoscyami ne , apoatropi ne , 6-hydroxyhyoscyami ne , scopolamine, 3,6-ditigloyl-7-oxytropane and metcloidine3 Britton-Robinson b u f f e r s (pH
.
1.8-8.0)
have f u r t h e r been a p p l i e d t o the separation o f codeine, quinine, dibazole, p l a t y p h y l l ine, hydrotartarate, s a l s o l ine, pachycarpine and papaverine9 Acetic
.
a c i d s o l u t i o n s o f d i f f e r e n t concentrations (0.88, 1.0, 4.4 and 5 m o l / l ) can also be ~ s e d ~ -Potassium ~ . hydrogen phthalate (0.05-0.10 m o l / l , pH 5,O) gives equally good r e s u l t s 8 . F i n a l l y borax (0.025 mol/l ; pH 9.2) has been used s u c ~ e s s f u l l y ~ * ~ , e.g.,
f o r ephedrine. P y r i d i n e acetate b u f f e r s (pH around 3.5) o r aqueous ammonia
(pH 11.0) a r e preferred i n t h e electrochromatography (mapping) o f d i v e r s e a1 kaloidsl'.
I n a systematic study o f anions used i n t h e e l e c t r o p h o r e t i c b u f f e r s i t
was found t h a t p e r c h l o r i c a c i d and t r i c h l o r o a c e t a t e are the anions o f choice when possible''. Separations are c a r r i e d a t about 20-25 V/cm; a running time 2 h i s u s u a l l y s u f f i c i e n t t o o b t a i n good separations. Some idea o f t h e m i g r a t i o n o f d i verse a l k a l o i d s i n an e l e c t r i c a l f i e l d can be gained from Tables 16.1-16.4. I n a d d i t i o n t o the vast number of r e s u l t s obtained on the separation o f alka-
l o i d s by paper electrophoresis , o t h e r sorbents have occasionally been used. Thus, polyacrylamide gel e l e c t r o p h ~ r e s i s ' ~i n acetate b u f f e r s was a p p l i e d t o t h e separat i o n o f raunatin and r e l a t e d compounds. Thin-layer e l e c t r o p h ~ r e s i s ' ~was made use o f by Wan using three d i f f e r e n t running b u f f e r s (Table 16.5). S i m i l a r r e s u l t s can be obtained a t lower voltages (500 V per p l a t e ) .
399 TABLE 16.5 ELECTROPHORETIC MOBILITIES OF ALKALOIDS AT 3000 V 15 cm x 20 cm p l a t e s coated w i t h C e l l u l o s e MN 300, t h i c k n e s s 0.5 mm15 A1 k a l o i d
.
Electrolyte Formic a c i d - a c e t i c a c i d water (26: 120: 1000)
Ammonia so1n.water (2:98)
MQa Atropine Brucine Cocaine Codeine Colchicine Emeti ne Ephedrine Ergometri ne Homatropine Hyo s c ine Mo r ph ine Papaverine Pilocarpine Physostigmine Quinidine Quin i ne Reserpine Strychnine Tubocurarine
.-
Ammonia s o l n w a t e r (10:90)
MAb
0.68 0.50 0.42 0.66 0.12 0.73 0.78 0.41 0.71 0.66 0.66 0.51 0.82 0.64 0.98 1.00 0.27 0.59 0.73
1.00 0.41 0.89 0.78 0.74 0.09 1.18 0.39 1.07 0.81 0.60 0.14 0.85 0.81 0.23 0.23 0.02 0.45 0.39
1.oo 0.42 0.90 0.81 0.74 0.11 1.16 0.40 1.10 0.83 0.58 0.17 0.84 0.82 0.23 0.24 0.00 0.45 0.37
1.00 0.42 0.83 0.77 0.68 0.10 1.09 0.37 1.04 0.80
-
0.16 0.81 0.77 0.22 0.23 0.03 0.43 0.29
1.00 0.40 0.83 0.76 0.83 0.11 1.13 0.35 1.04 0.86 -0.68 0.17 0.85 0.74 0.27 0.23 0.00 0.49 0.30
: M o b i l i t y r e l a t i v e t o q u i n i n e . E l e c t r o p h o r e t i c r u n : 10 min. M o b i l i t y r e l a t i v e t o a t r o p i n e . E l e c t r o p h o r e t i c r u n : 20 min. A l k a l o i d s s p o t t e d 3 cm fr m end o f p l a t e . 'As f o r b u t a l k a l o i d s s p o t t e d mid-way between ends o f p l a t e .
A l k a l o i d s e i t h e r absorb o f f l u o r e s c e i n UV l i g h t , which can be used f o r t h e i r d e t e c t i o n . The most f r e q u e n t d e t e c t i o n r e a g e n t s a r e cerium( I V ) s u l p h a t e , cinnamaldehyde-HC1
, Dragendorf
r e a g e n t , i o d o p l a t i n i c a c i d r e a g e n t or phosphomolybdic
a c i d r e a g e n t ( f o r a r e v i e w , see r e f . 16). REFERENCES
1 H.T.S. B r i t t o n and R.A. Robinson, J . Chern. Soc., (1931) 1456. 2 J.M. Calderwood and F. F i s h , J . Phurm. P h m a c o Z . , 2 1 (1969) 126. 3 S. Kisgyorgy, Rev. Med. (Tirgu-Mures R o n . ) , 17 (1971) 87. 4 G.C. C a s i n o r i , M. L e d e r e r and G.B. M a r i n i - B e t t d o , Guzz. Chim. I t a Z . , 86 (1956) 342.
400
5 C.L. Winek, J.L. Beal and M.P. Cava, J . Chromatogr., 10 (1963) 246. 6 G.B. M a r i n i - B e t t t i l o and M. L e d e r e r , Nature (London), 174 (1954) 133. 7 T. Kariyone, Y . Hashimoto, I . M o r i and M. Kimura, J . Pharm. Soc. J u p . , 73 (1953) 805. 8 H. i9ich1, J . Chrornutogr., 1 (1958) 93. 9 Nguyen-Hoang-Narn, M. H e r b e r t , Nguyen-Dat-Xuong and L . P i c h a t , B U Z Z . SOC. Chim. F r . , (1968) 2667. 10 J. G i s b e r t - C a l a b u i g , E. V i l l a n e u v a and G. Rubin de C e l i s , Zacchia, 6 (1970) 386. 11 M. S i n i b a l d i and B. R i n a l d u z z i , A m Z . L e t t . , 4 (1971) 125. 12 F. I n t r o n a , Med. Leg. A s s i c u r . , 12 (1964) 3. 13 G.B. M a r i n i - B e t t t i l o and J.A. Coch-Frugoni, &ZZ. Chim. I t a Z . , 8 (1956) 1324. 14 A.G. V o l l o s o v i c h , V . I . Safonov and M.P. Safonova, T r . Lenivqr. Khim.-Fum. I n s t . , 2 1 (1972) 192. 15 A.S.C. Wan, J . Chrornatogr., 60 (1971) 371. 16 G. Zweig and J.R. Whitaker, Paper Chrcmatography and EZectrophoresis, Academic Press, New York, 1961, p . 314.
Chapter 16 ALKALOIDS
2. DEYL A l k a l o i d s a r e n i t r o g e n o u s bases o f p l a n t o r i g i n and i n c l u d e a l m o s t a l l o f t h e known n i t r o g e n - c o n t a i n i n g r i n g systems. A g i v e n p l a n t s p e c i e s v e r y seldom c o n t a i n s a s i n g l e a l k a l o i d and u s u a l l y m i x t u r e s o f a l k a l o i d s w i t h s i m i l a r b i o l o g i c a l prope r t i e s a r e found t o g e t h e r . Commercial p r e p a r a t i o n s a r e u s u a l l y crude m i x t u r e s c0ntainin.g v a r y i n g amounts o f a l k a l o i d s and zone e l e c t r o p h o r e s i s o n paper has been w i d e l y a p p l i e d t o t h i s c a t e g o r y o f compounds. A1 k a l o i d s g e n e r a l l y m i g r a t e as c a t i o n s o v e r t h e pH range 1.8-12.5. TABLE 16.1 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS AT DIFFERENT pH VALUES (REF. 12)a A1 k a l o i d
A pomorph ine Atropine Caffeine Cocaine Ergotamine Heroin Lobe1 i n e Morphine Nicotine Papaveri ne Quinine Scopolamine Sparteine Strychnine Theobromine Tubocurarine a
M i g r a t i o n (cm) a t pH
1.8
2.1
-
8.4 12.4 - 3.9 - 14.0 - 7.5 - 13.7 - 12.5 - 13.8 - 24.3 - 11.4 - 17.9 - 11.9 - 20.1 - 11.8 - 4.4 - 15.0
10.0 14.1 - 5.9 14.6 6.8 - 13.6 - 12.6 - 14.0 27.8 - 12.6 - 20.2 - 14.0 - 22.1 - 13.4 - 4.3 - 14.9
-
-
-
-
-
4.2
-
7.2 14.0 - 5.1 - 16.3 - 7.2 - 15.2 - 14.2 - 15.3 - 18.3 - 12.1 - 14.9 - 13.0 - 18.8 - 12.6 - 5.2 15.3
-
.
6.4
-
5.2 14.3 - 6.4 14.5 - 4.5 - 12.7 - 12.7 - 13.1 17.2 - 7.6 - 11.5 - 14.6 - 15.8 - 12.1 - 6.7 - 12.7
-
-
-
8.6
-
4.2 15.2 8.0 - 14.7 - 6.2 12.0 - 13.2 - 12.0 - 13.0 - 5.0 10.2 - 11.6 - 16.8 - 10.5 - 8.4 11.8
-
-
-
-
-
11.2
-
2.0 13.7 - 8.5 9.9 - 4.4 - 7.0 - 4.4 - 7.2 - 7.5 - 5.1 - 7.2 8.7 - 16.7 - 8.0 - 6.1 - 8.3
-
-
-
12.5 1.0 - 6.0 - 3.5 - 10.1 - 6.3 - 7.6 - 7.4 - 7.9 - 3.5 - 3.0 - 3.0 - 4.0 - 9.5 - 3.0 - 6.5 - 9.0
B r i t t o n - R o b i n s o n phosphate-acetate-borate (0.04 m o l / l o f each) b u f f e r was used between pH 1.8 and 11.2 and a s o l u t i o n o f g l y c i n e a t pH 12.5; S c h l e i c h e r and S c h i i l l 2043 b paper ( 3 x 60 cm); 500 V ( 8 V/cm) f o r 3 h; movement i s towards t h e cathode except f o r apomorphine a t pH 12.5
396
TABLE 16.2 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS AT DIFFERENT pH VALUES (REF. 13)a ALKALOID
M i g r a t i o n (cm) i n b u f f e r b o f pH 2.3
Apomo r ph in e Arecol i n e Aspidospermine Atropine Berberine Bicuculline Bol d i n e Brucine Bul bocapnine Caffeine Cal ebassine Chel i d o n i n e C i n c h o n i d i ne Cinchonine Cocaine Col c h i c i n e Coniine C o r l umine Co r y d ine Curarine Cotarnine Dicentrine Dimet h y 1 b e r b e r ine Dinethyl tubocurarine Emetine Ephedrine Ergomet r ine Eserine F1 u o r o c u r i n e Gal eg ine Glaucine Harrnine Hero i n Homatropi ne Hydrast ine Hydrast in ine L-Hyoscyamine Jervine Lupan ine Lupinine Ma va c u r ine llescal i n e Mo r ph i ne Narceine N a r c o t i ne Nicotine Pa paver ine Pilocarpine P i per ine
- 4.2 - 8.8 - 5.24 - 6.3
- 1.5 - 5.1 - 2.2 - 4.5
-
- 3.7 - 0.3 - 6.5 - 4.95
10.4 10.2 6.1 0.2 8.9 4.95 4.6 10.6 8.4 1.5 6.6 6.8 7.5 8.0 3.5 6.15 7.7 9.1 2.8 @,.l 5.3 6.3 5.05 9.5 6.6 4.1 5.8 8.0 3.1 7.4 - 5.8 4.0 5.0 18.0 4.7 7.0 0
-
4.3
6.4
- 9.75 - 5.7 - 6.6 - 2.7 - 5.0 - 2.5 - 4.6 - 3.5 - 1.2 - 4.55 - 6.8 - 6.8 - 6.8 - 0.8 - 8.9 - 4.95 - 4.6 - 9.8 - 8.5 0 - 7.9 - 6.95 - 6.6 - 10.0 - 3.6 - 6.5 - 13.0 - 2.7 - 3.1 - 6.0 - 8.4 - 5.4 - 9.9 - 6.6 - 3.9 - 7.35 - 8.3 -
-
-
7.05 6.25 1.3 5.3 9.2 5.0 8.5
0
8.2
-. -13.5 8.7 10.7 - 2.0 6.2 3.0 6.9 3.3 2.35 8.0 5.8 9.3 8.0 - 11.0 2.1 13.0 5.9 7.1 11.0 12.5
-
-
-
0 - 10.4 - 9.9 - 8.5 - 10.0 - 3.8 - 10.5 - 8.0 - 11.8 - 3.8 2.6 - 9.5 - 10.4 - 6.1 - 13.2 - 10.3 - 5.3 - 10.6 - 12.1 - 2.4 - 10.2 - 10.0 - 2.6 - 5.4 - 12.3 - 3,o - 10.6 -
0.4
-
-
-
-
-
10.5 0 9.0 0 8.4 1.3 0 1.3 4.2 1.7 2.0
9.0
0 5.7 5.1 5.5 1.95 12.0 0 2.7 10.3 11.3 0 9.45 2.0 5.8 11.9 - 1.5 6.2 - 9.0 12.0 1.2 0 5.2 10.5 0 12.5 - 8.8 0 9.3 10.5 3.0 - 9.3 - 6.5 1.7 0 8.7
-
-
1.1 2.8 0 8.0 1.05 0 0 2.1 0.6 2.3 7.8 0 - 3.2 3.0 5.3 1.7 10.0 0 .- 1.4
-
-
-
-
-
-
-
--
-
10.6
- -1.3 - 5.0
- 10.0
4.0 0
-
-
10.8 0 7.8 8.7
10.8 - 0.3 0 2.85
0 - 11.3 6.9 0 8.7 10.4 2.4 8.2 2.85 - 1.65 0 - 4.5 0 1.95 0
-
0 1.5 0.9 0 3.8
-
-
-
0
-
-
-
-
-
-
-
-
-
-
-
11.4
-
-
-
-
-
1.3 0.3 5.0
0 0 0
1.5
-
2.1
-
0 6.4 4.5 0 5.3 4.9 0 2.2 1.5 0.9 or.2 6.8
0 0 0
1.5 0 4.5 1.8
r
1.8
- 1.3 - 1.9
-
1.2 1.3 1.5, 0 1.6 0 2.3 0
0.5
397 TABLE 16.2 (continued) ~~
.41k a l o i d
Protopine P r o t o v e r a t r i ne Quinine Sanquinarine Scopol ami ne Sempervirine Sol an ine Sparteine S t r y c hn ine Tetra hydropal ma t i n e Theobromine Theophyll i n e Tropacocaine Tropine Tubocurarine V e r a t r i ne Yohimbine
~
H i g r a t i o n (cm) i n b u f f e r b o f pH
2.3
4.3
6.4
8.2
- 4.6 - 3.5 - 10.2 - 1.5 - 6.7 - 0.8 - 2.65 - 11.5 - 5.6 - 4.5 - 0.45 - 0.25 - 6.7 - 7.5 - 7.0 - 4.0 - 4.5
- 5.0 - 3.5 - 6.8 - 1.38 - 6.65 - 0.7 - 2.9 - 10.0 - 5.85 - 4.2 - 0.8 - 1.0 - 8.6 - 6.6 - 6.7 - 3.7 - 4.7
- 7.7 - 5.85 - 6.5 - 4.8 - 7.6 - 4.6 - 1.1 0 - 10.4 - 6.0 - 0.5 0 - 4.0 - 4.0 - 10.8 - 10.1 - 8.5 - 5,2 - 4.1 0 - 2.1 - 1.8 - 2.1 0.9 - 10.2 - 10.6 - 14.5 - 5,85 - 8.4 - 5.8 - 6.2 - 3.8 - 6.8
10.5
11.4
- 3.9 - 2.5 0 - 3.3 -
0 0 0
-
0 0 9.9 0
0
1.3 0 0 4.7
--
0
- 1.8 1.15 - 8,8 0 - 4,7 .4 -- 11.0
0
2.0 5.5 2.0 0 0.7 0 0
aWhatman No. 1 paper; 8 V/cm f o r 3 h. bUniversal Britton-Robinson b u f f e r d i l u t e d 1:4. Movement i s towards t h e cathode i n most instances, as i n d i c a t e d by t h e minus signs.
TABLE 16.3 ELECTROPHORETIC MOBILITIES OF ALKALOIDS AT pH 5.0 AND 9.2 (REF. 8 ) A1 k a l o i d
Mobilitya i n b u f f e r o f DH 5.0b
Anabasi ne Anatabine N-Methylanabasine N-Methyl anatabine Metani c o t i n e Methylmetanicotine Myosmi ne
A1 k a l o i d
5.0b
9.2'
- 10.7 - 10.5 - 10.1 - 10.0 - 12.7 - 12.5 - 8.0 -
6.1 2.3 0.8 0.8 7.7 7.0 0.8
Mobilitya i n b u f f e r o f pH
Nicotine Nicotone N i co t y r i ne Nornicotine Norni c o t y r i ne Oxy nic o t ine
9.2'
-
12.1 - 3.4 - 3.2 - 12.2 - 1.5 - 3.9 -
;Mobility x lo5 cm2 V - l sec-' a t 10°C towards t h e cathode. Potassium hydrogen p h t h a l a t e b u f f e r (pH 5.0). Although t h k c o n c e n t r a t i o n o f b u f f e r i s n o t g i v e n i n r e f . 8, i t i s thought t h a t i t was 0.05 mol/l. '0.025 m o l / l borax s o l u t i o n (pH 9.2).
2.5 0.7 0
7.0 0.7 0.7
398
TABLE 16.4 ELECTROPHORETIC MIGRATIONS OF ALKALOIDS I N 5 mol/l ACETIC ACID' A1 k a l o i d
Migrationa (cm)
Aconi t i ne Atropine Bruci ne Caffeine Cinchoni ne Coni i n e Ephedrine
- 3.2 - 4.5 - 4.0 - 0.5
-
-
-
8.0 10.5 5.2
A1 k a l o i d
Migrationa (cm)
Hyoscyami ne Lobe1 ine N i co t i ne Quinine Sol ani ne Strychnine Sparteine
-
-
4.6 4.6 9.9 7.4 3.4 4.8 7.6
aMigration towards the cathode; 5 mol/l a c e t i c acid; 750 V f o r 2 h.
Universal s e r i e s o f Britton-Robinson b u f f e r s can be used f o r zone electropho1 r e s i s on paper. By aFplying a high voltage i t i s possible t o separate, e.g., candicine, tembetarine, N-methyl isocorydine and n i t i d i n e 2 Good separations a r e
.
obtained a t pH 8.0 w i t h l a u r i f o l i n e , magnoflorine, berberine, palmatine and t h e benzophenanthridines. A t t h i s pH i t i s also p o s s i b l e t o d i f f e r e n t i a t e between cuscohygri ne, a t r o p i n e , hyoscyami ne , apoatropi ne , 6-hydroxyhyoscyami ne , scopolamine, 3,6-ditigloyl-7-oxytropane and metcloidine3 Britton-Robinson b u f f e r s (pH
.
1.8-8.0)
have f u r t h e r been a p p l i e d t o the separation o f codeine, quinine, dibazole, p l a t y p h y l l ine, hydrotartarate, s a l s o l ine, pachycarpine and papaverine9 Acetic
.
a c i d s o l u t i o n s o f d i f f e r e n t concentrations (0.88, 1.0, 4.4 and 5 m o l / l ) can also be ~ s e d ~ -Potassium ~ . hydrogen phthalate (0.05-0.10 m o l / l , pH 5,O) gives equally good r e s u l t s 8 . F i n a l l y borax (0.025 mol/l ; pH 9.2) has been used s u c ~ e s s f u l l y ~ * ~ , e.g.,
f o r ephedrine. P y r i d i n e acetate b u f f e r s (pH around 3.5) o r aqueous ammonia
(pH 11.0) a r e preferred i n t h e electrochromatography (mapping) o f d i v e r s e a1 kaloidsl'.
I n a systematic study o f anions used i n t h e e l e c t r o p h o r e t i c b u f f e r s i t
was found t h a t p e r c h l o r i c a c i d and t r i c h l o r o a c e t a t e are the anions o f choice when possible''. Separations are c a r r i e d a t about 20-25 V/cm; a running time 2 h i s u s u a l l y s u f f i c i e n t t o o b t a i n good separations. Some idea o f t h e m i g r a t i o n o f d i verse a l k a l o i d s i n an e l e c t r i c a l f i e l d can be gained from Tables 16.1-16.4. I n a d d i t i o n t o the vast number of r e s u l t s obtained on the separation o f alka-
l o i d s by paper electrophoresis , o t h e r sorbents have occasionally been used. Thus, polyacrylamide gel e l e c t r o p h ~ r e s i s ' ~i n acetate b u f f e r s was a p p l i e d t o t h e separat i o n o f raunatin and r e l a t e d compounds. Thin-layer e l e c t r o p h ~ r e s i s ' ~was made use o f by Wan using three d i f f e r e n t running b u f f e r s (Table 16.5). S i m i l a r r e s u l t s can be obtained a t lower voltages (500 V per p l a t e ) .
399 TABLE 16.5 ELECTROPHORETIC MOBILITIES OF ALKALOIDS AT 3000 V 15 cm x 20 cm p l a t e s coated w i t h C e l l u l o s e MN 300, t h i c k n e s s 0.5 mm15 A1 k a l o i d
.
Electrolyte Formic a c i d - a c e t i c a c i d water (26: 120: 1000)
Ammonia so1n.water (2:98)
MQa Atropine Brucine Cocaine Codeine Colchicine Emeti ne Ephedrine Ergometri ne Homatropine Hyo s c ine Mo r ph ine Papaverine Pilocarpine Physostigmine Quinidine Quin i ne Reserpine Strychnine Tubocurarine
.-
Ammonia s o l n w a t e r (10:90)
MAb
0.68 0.50 0.42 0.66 0.12 0.73 0.78 0.41 0.71 0.66 0.66 0.51 0.82 0.64 0.98 1.00 0.27 0.59 0.73
1.00 0.41 0.89 0.78 0.74 0.09 1.18 0.39 1.07 0.81 0.60 0.14 0.85 0.81 0.23 0.23 0.02 0.45 0.39
1.oo 0.42 0.90 0.81 0.74 0.11 1.16 0.40 1.10 0.83 0.58 0.17 0.84 0.82 0.23 0.24 0.00 0.45 0.37
1.00 0.42 0.83 0.77 0.68 0.10 1.09 0.37 1.04 0.80
-
0.16 0.81 0.77 0.22 0.23 0.03 0.43 0.29
1.00 0.40 0.83 0.76 0.83 0.11 1.13 0.35 1.04 0.86 -0.68 0.17 0.85 0.74 0.27 0.23 0.00 0.49 0.30
: M o b i l i t y r e l a t i v e t o q u i n i n e . E l e c t r o p h o r e t i c r u n : 10 min. M o b i l i t y r e l a t i v e t o a t r o p i n e . E l e c t r o p h o r e t i c r u n : 20 min. A l k a l o i d s s p o t t e d 3 cm fr m end o f p l a t e . 'As f o r b u t a l k a l o i d s s p o t t e d mid-way between ends o f p l a t e .
A l k a l o i d s e i t h e r absorb o f f l u o r e s c e i n UV l i g h t , which can be used f o r t h e i r d e t e c t i o n . The most f r e q u e n t d e t e c t i o n r e a g e n t s a r e cerium( I V ) s u l p h a t e , cinnamaldehyde-HC1
, Dragendorf
r e a g e n t , i o d o p l a t i n i c a c i d r e a g e n t or phosphomolybdic
a c i d r e a g e n t ( f o r a r e v i e w , see r e f . 16). REFERENCES
1 H.T.S. B r i t t o n and R.A. Robinson, J . Chern. Soc., (1931) 1456. 2 J.M. Calderwood and F. F i s h , J . Phurm. P h m a c o Z . , 2 1 (1969) 126. 3 S. Kisgyorgy, Rev. Med. (Tirgu-Mures R o n . ) , 17 (1971) 87. 4 G.C. C a s i n o r i , M. L e d e r e r and G.B. M a r i n i - B e t t d o , Guzz. Chim. I t a Z . , 86 (1956) 342.
400
5 C.L. Winek, J.L. Beal and M.P. Cava, J . Chromatogr., 10 (1963) 246. 6 G.B. M a r i n i - B e t t t i l o and M. L e d e r e r , Nature (London), 174 (1954) 133. 7 T. Kariyone, Y . Hashimoto, I . M o r i and M. Kimura, J . Pharm. Soc. J u p . , 73 (1953) 805. 8 H. i9ich1, J . Chrornutogr., 1 (1958) 93. 9 Nguyen-Hoang-Narn, M. H e r b e r t , Nguyen-Dat-Xuong and L . P i c h a t , B U Z Z . SOC. Chim. F r . , (1968) 2667. 10 J. G i s b e r t - C a l a b u i g , E. V i l l a n e u v a and G. Rubin de C e l i s , Zacchia, 6 (1970) 386. 11 M. S i n i b a l d i and B. R i n a l d u z z i , A m Z . L e t t . , 4 (1971) 125. 12 F. I n t r o n a , Med. Leg. A s s i c u r . , 12 (1964) 3. 13 G.B. M a r i n i - B e t t t i l o and J.A. Coch-Frugoni, &ZZ. Chim. I t a Z . , 8 (1956) 1324. 14 A.G. V o l l o s o v i c h , V . I . Safonov and M.P. Safonova, T r . Lenivqr. Khim.-Fum. I n s t . , 2 1 (1972) 192. 15 A.S.C. Wan, J . Chrornatogr., 60 (1971) 371. 16 G. Zweig and J.R. Whitaker, Paper Chrcmatography and EZectrophoresis, Academic Press, New York, 1961, p . 314.