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Analytical problems in the determination of photoproducts of hydroxamic acids
The Science of the Total Environment, 67 (1987) 69-74 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
69
A N A L Y T I C A L P R O B L E M S IN THE D E T E R M I N A T I O N OF P H O T O P R O D U C T S OF H Y D R O X A M I C A C I D S *
JAN KOCHANY Institute for Environmental Protection, ul. Krucza 5/11, 00-548 Warsaw (Poland)
EWA LIPCZYNSKA-KOCHANY Warsaw Technical University, Department of Chemistry, ul. Koszykowa 75, 00-662 Warsaw (Poland)
(Received March 22nd, 1987; accepted April 24th, 1987)
ABSTRACT Results of studies of the photodegradation of aromatic hydroxamic acids, to compounds suspected to be carcinogenic and mutagenic, have stimulated an interest in the analysis of these acids and their photoproducts. In this paper, results are reported of investigations on the influence of phosphate buffer concentration, buffer]methanol ratio and eluent pH on the separation of photoproducts formed after irradiation of N-phenyl-benzenecarbohydroxamic, N-methyl-2-naphthalenecarbohydroxamic and N-/p-tolyl/-2-naphthalenecarbohydroxamic acids. The best separation conditions were achieved using a 0.1 M buffer concentration and an eluent pHe of 4.0-5.2. At a pHe lower than 3.5, on-column hydrolysis of the hydroxamic acids was observed. INTRODUCTION H y d r o x a m i c acids s h o w a wide s p e c t r u m of b i o l o g i c a l a c t i v i t y [1]: n a t u r a l l y o c c u r r i n g h y d r o x a m i c acids, p r o d u c e d m a i n l y by fungi, a r e a c t i v e as antibiotics, a n t i t u m o r a g e n t s , f u n g i s t a t i c s , a n d g r o w t h a n d cell division f a c t o r s [2]. T h e y also p l a y a n i m p o r t a n t role in i r o n u p t a k e a n d m e t a b o l i s m [2]. M a n y s y n t h e t i c h y d r o x a m i c acids s h o w fungicidal, a n t i m a l a r i a l a n d antib a c t e r i a l a c t i v i t i e s a n d h a v e f o u n d t h e r a p e u t i c a p p l i c a t i o n s [3, 4]. S o m e hydr o x a m i c acids i n h i b i t r i b o n u c l e o t i d e r e d u c t a s e , a n i r o n - r e q u i r i n g e n z y m e esse n t i a l for D N A b i o s y n t h e s i s [5]. O t h e r s y n t h e t i c acids h a v e b e e n r e p o r t e d to be a c t i v e as pesticides, to p r o m o t e t h e g r o w t h of p l a n t s a n d to i m p r o v e soil q u a l i t y [6]. On t h e o t h e r h a n d , r e s u l t s of r e c e n t studies h a v e r e v e a l e d t h a t m a n y hydr o x a m i c acids a r e p o w e r f u l m u t a g e n s [7] a n d c a r c i n o g e n s [8]. As m a n y n a t u r a l a n d s y n t h e t i c h y d r o x a m i c acids o c c u r in t h e e n v i r o n m e n t a n d a r e e x p o s e d to s u n l i g h t , it is i m p o r t a n t to s t u d y t h e i r p h o t o s t a b i l i t y a n d i d e n t i f y possible p h o t o d e c o m p o s i t i o n p r o d u c t s . * Chemistry of Hydroxamic Acids, Part 13. For Part 12 see; E. Lipczyfska-Kochany and J. Kochany, J. Photochem., 38 (1987) 331-343.
0048-9697/87/$03.50
© 1987 Elsevier Science Publishers B.V.
70 Preliminary studies of the photochemistry of N-phenyl-benzenecarbohydroxamic acid [9] and naphthalenecarbohydroxamic acids [10] have revealed that hydroxamic acids are photolabile. These results therefore suggest that the photoirradiation of hydroxamic acids may lead to the formation of toxic and/or carcinogenic photoproducts and this has stimulated interest in their analysis. Results of preliminary studies have shown [11, 12] that the "ion suppression technique" [13] can be used successfully to analyse some hydroxamic acids. Difficulties in separating the complex mixtures, obtained after the photoirradiation of aromatic hydroxamic acids [9, 10], prompted us to undertake more detailed studies on the subject. In this paper we report the results of our investigations on the influence of phosphate buffer concentration, buffer/methanol ratio and eluent pH on peak shape and separation of photoproducts after the irradiation of N-phenylbenzenecarbohydroxamic, N-methyl-2-napthalenecarbohydroxamic and N-/ptolyl/-2-naphthalenecarbohydroxamic acids. EXPERIMENTAL
NoPhenyl-benzenecarbohydroxamic acid was obtained by the reaction of N-phenylhydroxylamine with benzoyl chloride, and N-methyl-2-naphthalenecarbohydroxamic and N-/p-tolyl/-2-naphthalenecarbohydroxamic acids were prepared by the reactions of 2-naphthoyl chloride with N-methylhydroxylamine and N-/p-tolyl/-hydroxylamine, respectively. These products gave satis-
Ar/H'I' 1.2
1
0.9
/
2 i/3//
o.~
2.0
~/./
4.0
6.0 PHe
Fig. 1. Dependence of the peak shape, defined as Ar/HT, of (1,
) N-phenyl-benzene-
c a r b o h y d r o x a m i c acid; (2, - - - ) N - m e t h y l - 2 - n a p h t h a l e n e c a r b o h y d r o x a m i c acid; and (3, - . . . . ) N-/pt o l y l / - 2 - n a p h t h a l e n e c a r b o h y d r o x a m i c acid on t h e mobile phase pH value (pile): Ar = peak area, HT = peak height.
71 A [%]
6.0
5.0
4,0 ~'~,. 1
,\ ',\.\
3.0
"
2.0
"
2
,, .\.
~' %%'e
2,0
3.0
4.0
5.0 PH e
Fig. 2. Percentage of hydroxamic acid hydrolysed (A) as a function of the pH of the mobile phase (pile). (1, - . . . . ) N./p.tolyl/-2-naphthalenecarbohydroxamic acid, and (2, - - - ) N-methyl-2naphthalenecarbohydroxamic acid.
factory elemental analyses and their physicochemical properties agreed well with their structures. Details of the synthesis and photochemistry of the compounds are reported elsewhere [9, 10]. All reagents used in this work were purchased from E. Merck (Darmstadt, G.F.R.). Chromatographic analyses were performed on a PYE UNICAM Isocratic Liquid Chromatograph, Model 4000, equipped with a PU 4020 variable UV detector, operated at ~ = 260 or 280 nm. A stainless steel 250 x 4.6mm i.d. column packed with Lichrosorb-ODS, 5/~m average particle size (E. Merck, Darmstadt, G.F.R.), was used with eluent flow rates of 1-2 ml min-1. The column temperature was not thermostatically controlled but generally kept at about 20°C. Mobile phases were prepared by mixing methanol with 0.01 M, 0.05 M and 0.1 M phosphate buffer over the ratio range 1:3 to 3:1. The phosphate buffer was obtained by dissolving disodium hydrogen phosphate (E. Merck, G.F.R.) in redistilled water and adjusting the pH to 3.5 with phosphoric acid (E. Merck, Suprapur). The mixed solvents were filtered through membranes (Acro LC 13, 0.45/~m, Gelman Sciences, U.S.A.) and degassed using helium (Air Products, U.S.A.). The solvent peak was used as the reference for dead time determination. RESULTS AND DISCUSSION
We studied the effect of the phosphate buffer concentration (pH = 3.5, buffer/methanol = 1:1) on peak shape and found that dilution of 0.1 M buffer
72 k 8.0
7.0 1
6.0
2
5.0 3
4.0
4.,,
3.0
\ \ ~ " " "-.... 5
2.0
1.O
t
I 1.0
t
l 2.0
i
I 3.0
met han°~bu f fer pH =3.5
Fig. 3. Dependence of the capacity ratios (k) of photoproducts, after N-phenyl-benzenecarbohydroxamic acid irradiation, on the methanol/buffer (0.1 M, pH = 3.5) ratio; a Lichrosorb ODS 5/~m column was used. (1, o o) Benzanilide; (2, h A) benzaldehyde; (3,-s-s-) N-phenylbenzenecarbamate; (4, - . - ) N-phenylbenzenecarbohydroxamic acid; (5, - - - ) N-phenylhydroxylamine; (6, ) benzoic acid.
to between 0.05 and 0.01 M leads to an increase in peak width while the retention times of the hydroxamic acids remained almost unaffected. Increasing the buffer concentration from about 0.1 M to saturation does not improve peak shape and can harm the pump. Studies of quantum yields of the photoreactions of the compounds under consideration require very accurate quantitative determination of the concentrations of the hydroxamic acids. In a search for the best conditions which would give sharp chromatographic peaks, we investigated the effect of the pH of the mobile phase (pile). As can be seen from Fig. 1, variations over the range
73 pile = 2.5-5.2 do n o t h a v e a m a j o r c h r o m a t o g r a p h i c effect, while a n i n c r e a s e of pile a b o v e 5.2 r e s u l t s in c h a n g e s in p e a k width. We also studied t h e s t a b i l i t y of N - m e t h y l - 2 - n a p h t h a l e n e c a r b o h y d r o x a m i c a n d N./p.tolyl/-2-naphthalenecarbohydroxamicacids, in b u f f e r / m e t h a n o l mixtures, as t h e y p a s s e d t h r o u g h t h e c h r o m a t o g r a p h i c column. W e found t h a t t h e c o m p o u n d s h y d r o l y z e w h e n t h e e l u e n t pH~ is a b o u t 3.8 or below. T h i s h a s a s i g n i f i c a n t i n f l u e n c e on t h e a n a l y t i c a l r e s u l t s o b t a i n e d w i t h mobile p h a s e s o f pH~ l o w e r t h a n 2 (Fig. 2). T h u s , pile = 4.0-5.2 w a s c h o s e n as b e i n g t h e m o s t useful for o u r purposes, as PIle v a l u e s l o w e r t h a n 4.0 t h r e a t e n e d t h e s t a b i l i t y of t h e h y d r o x a m i c acids u n d e r study. I n o r d e r to find t h e b e s t s e p a r a t i o n of p h o t o p r o d u c t s , a n a l y s e s u s i n g diff e r e n t m e t h a n o l / b u f f e r r a t i o s w e r e p e r f o r m e d w i t h a buffer c o n c e n t r a t i o n of 0.1 M a n d p H = 3.5. C h a n g e s in t h e c a p a c i t y ratio, (k), v e r s u s m e t h a n o l / b u f f e r r a t i o o b t a i n e d for N - p h e n y l - b e n z e n e c a r b o h y d r o x a m i c acid a n d its photop r o d u c t s a r e s h o w n in Fig. 3. S i m i l a r b e h a v i o u r w a s o b s e r v e d for t h e o t h e r h y d r o x a m i c acids u n d e r c o n s i d e r a t i o n . I n c r e a s i n g t h e a m o u n t of b u f f e r in t h e m o b i l e p h a s e i m p r o v e s t h e s e p a r a t i o n , a n d s a t i s f a c t o r y r e s u l t s a r e o b t a i n e d w h e n t h e r a t i o is k e p t b e t w e e n 0.2 a n d 2.0. H o w e v e r , it is i m p o r t a n t to n o t e t h a t small c h a n g e s of e l u e n t c o m p o n e n t r a t i o (e.g. 1.4 to 1.7 in t h e c a s e of N - p h e n y l - b e n z e n e c a r b o h y d r o x a m i c acid) c a n a d v e r s e l y affect t h e s e p a r a t i o n of s o m e p h o t o p r o d u c t s a n d e v e n c h a n g e the s e q u e n c e of elution. I n c o n c l u s i o n , t h e ~'ion s u p p r e s s i o n t e c h n i q u e " c a n be r e c o m m e n d e d as a n e x c e l l e n t m e t h o d for t h e a n a l y s i s of h y d r o x a m i c acids a n d t h e i r p h o t o p r o d u c t s . T h e s o l v e n t c o m p o s i t i o n , pile a n d buffer c o n c e n t r a t i o n m u s t be r i g o r o u s l y c o n t r o l l e d to e n s u r e r e p r o d u c i b i l i t y . REFERENCES 1 H. Kehl (Ed.), Chemistry and Biology of Hydroxamic Acids, Karger, New York, 1982. 2 J.B. Neilands, Hydroxamic acids in nature, Science, 156 (1967) 1443-1447. 3 N.P. Buu-Hoi, G. Lameblin, C. Lepoivre, C. Gillet, M. Gautier and J. Thiriaux, un nouvel agent antiinflammatoire de structure non-steroidique; L'acide p-butoxyphenylacetohydroxamicique, Compt. Rend., 261 (1965) 2259-2263. 4 N.R. Gevirtz, D. Tendler, G. Lurinsky and L.R. Wasserman, Clinical studies on storage iron with desferrioxamine, N. Engl. J. Med., 273 (1965) 95-97. 5 B. Van't Riet, L.B. Kier and H.L. Elford, Structure-activity relationship of benzohydroxamic acid inhibitors of ribonucleotide reductase, J. Pharm. Sci., 69 (1980) 856-857. 6 L.S. Waid, in E.A. Paul and A.D. McLaren (Eds.), Hydroxamic Acids in Soil Systems, Soil Biochem., Marcel Dekker, New York, 1975 pp. 65-101. 7 E. LipczySska-Kochany, H. Iwamura, K. Takahashi, A. Hakura and Y. Kawazoe, Mutagenicity of pyridine- and quinolinecarbohydroxamic acids derivatives, Mutat. Res., 135 (1894) 139-148. 8 D. Malejka-Giganti, Carcinogenicity of N-Arylhydroxamic Acids, in H. Kehl (Ed.), Chemistry and Biology of Hydroxamic Acids, Karger, New York, 1982, pp. 149-160. 9 E. LipczySska-Kochany and J. Kochany, Photochemistry of N-phenyl-benzenecarbohydroxamic acids. Studies on the mechanism of photoreaction, J. Photochem., 38 (1987) 331-343.
74 10 11
12 13
E. Lipczyfiska-Kochany and J. Kochany, Photochemistry of Naphthalenecarbohydroxamic acids, in preparation. A.J. Barnicoat, W.G. Van't Hoff, P.J. Morrison and I.D. Bradbrook, Determination of salicylhydroxamic acid, a trypanocidal agent, by reversed-phase high-performance liquid chromatography, J. Chromatogr., 225 (1981) 23~239. E. Lipczyfiska-Kochany, High performance liquid chromatographic analysis of naphthaleneand anthranilohydroxamic acids, J. Chromatogr., 260 (1983) 493-496. B.A. Bidlingmeyer, Separation of ionic compounds by reversed-phase liquid chromatography: an update of ion-pairing techniques, J. Chromatogr. Sci., 18 (1980) 525-539.
69
A N A L Y T I C A L P R O B L E M S IN THE D E T E R M I N A T I O N OF P H O T O P R O D U C T S OF H Y D R O X A M I C A C I D S *
JAN KOCHANY Institute for Environmental Protection, ul. Krucza 5/11, 00-548 Warsaw (Poland)
EWA LIPCZYNSKA-KOCHANY Warsaw Technical University, Department of Chemistry, ul. Koszykowa 75, 00-662 Warsaw (Poland)
(Received March 22nd, 1987; accepted April 24th, 1987)
ABSTRACT Results of studies of the photodegradation of aromatic hydroxamic acids, to compounds suspected to be carcinogenic and mutagenic, have stimulated an interest in the analysis of these acids and their photoproducts. In this paper, results are reported of investigations on the influence of phosphate buffer concentration, buffer]methanol ratio and eluent pH on the separation of photoproducts formed after irradiation of N-phenyl-benzenecarbohydroxamic, N-methyl-2-naphthalenecarbohydroxamic and N-/p-tolyl/-2-naphthalenecarbohydroxamic acids. The best separation conditions were achieved using a 0.1 M buffer concentration and an eluent pHe of 4.0-5.2. At a pHe lower than 3.5, on-column hydrolysis of the hydroxamic acids was observed. INTRODUCTION H y d r o x a m i c acids s h o w a wide s p e c t r u m of b i o l o g i c a l a c t i v i t y [1]: n a t u r a l l y o c c u r r i n g h y d r o x a m i c acids, p r o d u c e d m a i n l y by fungi, a r e a c t i v e as antibiotics, a n t i t u m o r a g e n t s , f u n g i s t a t i c s , a n d g r o w t h a n d cell division f a c t o r s [2]. T h e y also p l a y a n i m p o r t a n t role in i r o n u p t a k e a n d m e t a b o l i s m [2]. M a n y s y n t h e t i c h y d r o x a m i c acids s h o w fungicidal, a n t i m a l a r i a l a n d antib a c t e r i a l a c t i v i t i e s a n d h a v e f o u n d t h e r a p e u t i c a p p l i c a t i o n s [3, 4]. S o m e hydr o x a m i c acids i n h i b i t r i b o n u c l e o t i d e r e d u c t a s e , a n i r o n - r e q u i r i n g e n z y m e esse n t i a l for D N A b i o s y n t h e s i s [5]. O t h e r s y n t h e t i c acids h a v e b e e n r e p o r t e d to be a c t i v e as pesticides, to p r o m o t e t h e g r o w t h of p l a n t s a n d to i m p r o v e soil q u a l i t y [6]. On t h e o t h e r h a n d , r e s u l t s of r e c e n t studies h a v e r e v e a l e d t h a t m a n y hydr o x a m i c acids a r e p o w e r f u l m u t a g e n s [7] a n d c a r c i n o g e n s [8]. As m a n y n a t u r a l a n d s y n t h e t i c h y d r o x a m i c acids o c c u r in t h e e n v i r o n m e n t a n d a r e e x p o s e d to s u n l i g h t , it is i m p o r t a n t to s t u d y t h e i r p h o t o s t a b i l i t y a n d i d e n t i f y possible p h o t o d e c o m p o s i t i o n p r o d u c t s . * Chemistry of Hydroxamic Acids, Part 13. For Part 12 see; E. Lipczyfska-Kochany and J. Kochany, J. Photochem., 38 (1987) 331-343.
0048-9697/87/$03.50
© 1987 Elsevier Science Publishers B.V.
70 Preliminary studies of the photochemistry of N-phenyl-benzenecarbohydroxamic acid [9] and naphthalenecarbohydroxamic acids [10] have revealed that hydroxamic acids are photolabile. These results therefore suggest that the photoirradiation of hydroxamic acids may lead to the formation of toxic and/or carcinogenic photoproducts and this has stimulated interest in their analysis. Results of preliminary studies have shown [11, 12] that the "ion suppression technique" [13] can be used successfully to analyse some hydroxamic acids. Difficulties in separating the complex mixtures, obtained after the photoirradiation of aromatic hydroxamic acids [9, 10], prompted us to undertake more detailed studies on the subject. In this paper we report the results of our investigations on the influence of phosphate buffer concentration, buffer/methanol ratio and eluent pH on peak shape and separation of photoproducts after the irradiation of N-phenylbenzenecarbohydroxamic, N-methyl-2-napthalenecarbohydroxamic and N-/ptolyl/-2-naphthalenecarbohydroxamic acids. EXPERIMENTAL
NoPhenyl-benzenecarbohydroxamic acid was obtained by the reaction of N-phenylhydroxylamine with benzoyl chloride, and N-methyl-2-naphthalenecarbohydroxamic and N-/p-tolyl/-2-naphthalenecarbohydroxamic acids were prepared by the reactions of 2-naphthoyl chloride with N-methylhydroxylamine and N-/p-tolyl/-hydroxylamine, respectively. These products gave satis-
Ar/H'I' 1.2
1
0.9
/
2 i/3//
o.~
2.0
~/./
4.0
6.0 PHe
Fig. 1. Dependence of the peak shape, defined as Ar/HT, of (1,
) N-phenyl-benzene-
c a r b o h y d r o x a m i c acid; (2, - - - ) N - m e t h y l - 2 - n a p h t h a l e n e c a r b o h y d r o x a m i c acid; and (3, - . . . . ) N-/pt o l y l / - 2 - n a p h t h a l e n e c a r b o h y d r o x a m i c acid on t h e mobile phase pH value (pile): Ar = peak area, HT = peak height.
71 A [%]
6.0
5.0
4,0 ~'~,. 1
,\ ',\.\
3.0
"
2.0
"
2
,, .\.
~' %%'e
2,0
3.0
4.0
5.0 PH e
Fig. 2. Percentage of hydroxamic acid hydrolysed (A) as a function of the pH of the mobile phase (pile). (1, - . . . . ) N./p.tolyl/-2-naphthalenecarbohydroxamic acid, and (2, - - - ) N-methyl-2naphthalenecarbohydroxamic acid.
factory elemental analyses and their physicochemical properties agreed well with their structures. Details of the synthesis and photochemistry of the compounds are reported elsewhere [9, 10]. All reagents used in this work were purchased from E. Merck (Darmstadt, G.F.R.). Chromatographic analyses were performed on a PYE UNICAM Isocratic Liquid Chromatograph, Model 4000, equipped with a PU 4020 variable UV detector, operated at ~ = 260 or 280 nm. A stainless steel 250 x 4.6mm i.d. column packed with Lichrosorb-ODS, 5/~m average particle size (E. Merck, Darmstadt, G.F.R.), was used with eluent flow rates of 1-2 ml min-1. The column temperature was not thermostatically controlled but generally kept at about 20°C. Mobile phases were prepared by mixing methanol with 0.01 M, 0.05 M and 0.1 M phosphate buffer over the ratio range 1:3 to 3:1. The phosphate buffer was obtained by dissolving disodium hydrogen phosphate (E. Merck, G.F.R.) in redistilled water and adjusting the pH to 3.5 with phosphoric acid (E. Merck, Suprapur). The mixed solvents were filtered through membranes (Acro LC 13, 0.45/~m, Gelman Sciences, U.S.A.) and degassed using helium (Air Products, U.S.A.). The solvent peak was used as the reference for dead time determination. RESULTS AND DISCUSSION
We studied the effect of the phosphate buffer concentration (pH = 3.5, buffer/methanol = 1:1) on peak shape and found that dilution of 0.1 M buffer
72 k 8.0
7.0 1
6.0
2
5.0 3
4.0
4.,,
3.0
\ \ ~ " " "-.... 5
2.0
1.O
t
I 1.0
t
l 2.0
i
I 3.0
met han°~bu f fer pH =3.5
Fig. 3. Dependence of the capacity ratios (k) of photoproducts, after N-phenyl-benzenecarbohydroxamic acid irradiation, on the methanol/buffer (0.1 M, pH = 3.5) ratio; a Lichrosorb ODS 5/~m column was used. (1, o o) Benzanilide; (2, h A) benzaldehyde; (3,-s-s-) N-phenylbenzenecarbamate; (4, - . - ) N-phenylbenzenecarbohydroxamic acid; (5, - - - ) N-phenylhydroxylamine; (6, ) benzoic acid.
to between 0.05 and 0.01 M leads to an increase in peak width while the retention times of the hydroxamic acids remained almost unaffected. Increasing the buffer concentration from about 0.1 M to saturation does not improve peak shape and can harm the pump. Studies of quantum yields of the photoreactions of the compounds under consideration require very accurate quantitative determination of the concentrations of the hydroxamic acids. In a search for the best conditions which would give sharp chromatographic peaks, we investigated the effect of the pH of the mobile phase (pile). As can be seen from Fig. 1, variations over the range
73 pile = 2.5-5.2 do n o t h a v e a m a j o r c h r o m a t o g r a p h i c effect, while a n i n c r e a s e of pile a b o v e 5.2 r e s u l t s in c h a n g e s in p e a k width. We also studied t h e s t a b i l i t y of N - m e t h y l - 2 - n a p h t h a l e n e c a r b o h y d r o x a m i c a n d N./p.tolyl/-2-naphthalenecarbohydroxamicacids, in b u f f e r / m e t h a n o l mixtures, as t h e y p a s s e d t h r o u g h t h e c h r o m a t o g r a p h i c column. W e found t h a t t h e c o m p o u n d s h y d r o l y z e w h e n t h e e l u e n t pH~ is a b o u t 3.8 or below. T h i s h a s a s i g n i f i c a n t i n f l u e n c e on t h e a n a l y t i c a l r e s u l t s o b t a i n e d w i t h mobile p h a s e s o f pH~ l o w e r t h a n 2 (Fig. 2). T h u s , pile = 4.0-5.2 w a s c h o s e n as b e i n g t h e m o s t useful for o u r purposes, as PIle v a l u e s l o w e r t h a n 4.0 t h r e a t e n e d t h e s t a b i l i t y of t h e h y d r o x a m i c acids u n d e r study. I n o r d e r to find t h e b e s t s e p a r a t i o n of p h o t o p r o d u c t s , a n a l y s e s u s i n g diff e r e n t m e t h a n o l / b u f f e r r a t i o s w e r e p e r f o r m e d w i t h a buffer c o n c e n t r a t i o n of 0.1 M a n d p H = 3.5. C h a n g e s in t h e c a p a c i t y ratio, (k), v e r s u s m e t h a n o l / b u f f e r r a t i o o b t a i n e d for N - p h e n y l - b e n z e n e c a r b o h y d r o x a m i c acid a n d its photop r o d u c t s a r e s h o w n in Fig. 3. S i m i l a r b e h a v i o u r w a s o b s e r v e d for t h e o t h e r h y d r o x a m i c acids u n d e r c o n s i d e r a t i o n . I n c r e a s i n g t h e a m o u n t of b u f f e r in t h e m o b i l e p h a s e i m p r o v e s t h e s e p a r a t i o n , a n d s a t i s f a c t o r y r e s u l t s a r e o b t a i n e d w h e n t h e r a t i o is k e p t b e t w e e n 0.2 a n d 2.0. H o w e v e r , it is i m p o r t a n t to n o t e t h a t small c h a n g e s of e l u e n t c o m p o n e n t r a t i o (e.g. 1.4 to 1.7 in t h e c a s e of N - p h e n y l - b e n z e n e c a r b o h y d r o x a m i c acid) c a n a d v e r s e l y affect t h e s e p a r a t i o n of s o m e p h o t o p r o d u c t s a n d e v e n c h a n g e the s e q u e n c e of elution. I n c o n c l u s i o n , t h e ~'ion s u p p r e s s i o n t e c h n i q u e " c a n be r e c o m m e n d e d as a n e x c e l l e n t m e t h o d for t h e a n a l y s i s of h y d r o x a m i c acids a n d t h e i r p h o t o p r o d u c t s . T h e s o l v e n t c o m p o s i t i o n , pile a n d buffer c o n c e n t r a t i o n m u s t be r i g o r o u s l y c o n t r o l l e d to e n s u r e r e p r o d u c i b i l i t y . REFERENCES 1 H. Kehl (Ed.), Chemistry and Biology of Hydroxamic Acids, Karger, New York, 1982. 2 J.B. Neilands, Hydroxamic acids in nature, Science, 156 (1967) 1443-1447. 3 N.P. Buu-Hoi, G. Lameblin, C. Lepoivre, C. Gillet, M. Gautier and J. Thiriaux, un nouvel agent antiinflammatoire de structure non-steroidique; L'acide p-butoxyphenylacetohydroxamicique, Compt. Rend., 261 (1965) 2259-2263. 4 N.R. Gevirtz, D. Tendler, G. Lurinsky and L.R. Wasserman, Clinical studies on storage iron with desferrioxamine, N. Engl. J. Med., 273 (1965) 95-97. 5 B. Van't Riet, L.B. Kier and H.L. Elford, Structure-activity relationship of benzohydroxamic acid inhibitors of ribonucleotide reductase, J. Pharm. Sci., 69 (1980) 856-857. 6 L.S. Waid, in E.A. Paul and A.D. McLaren (Eds.), Hydroxamic Acids in Soil Systems, Soil Biochem., Marcel Dekker, New York, 1975 pp. 65-101. 7 E. LipczySska-Kochany, H. Iwamura, K. Takahashi, A. Hakura and Y. Kawazoe, Mutagenicity of pyridine- and quinolinecarbohydroxamic acids derivatives, Mutat. Res., 135 (1894) 139-148. 8 D. Malejka-Giganti, Carcinogenicity of N-Arylhydroxamic Acids, in H. Kehl (Ed.), Chemistry and Biology of Hydroxamic Acids, Karger, New York, 1982, pp. 149-160. 9 E. LipczySska-Kochany and J. Kochany, Photochemistry of N-phenyl-benzenecarbohydroxamic acids. Studies on the mechanism of photoreaction, J. Photochem., 38 (1987) 331-343.
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E. Lipczyfiska-Kochany and J. Kochany, Photochemistry of Naphthalenecarbohydroxamic acids, in preparation. A.J. Barnicoat, W.G. Van't Hoff, P.J. Morrison and I.D. Bradbrook, Determination of salicylhydroxamic acid, a trypanocidal agent, by reversed-phase high-performance liquid chromatography, J. Chromatogr., 225 (1981) 23~239. E. Lipczyfiska-Kochany, High performance liquid chromatographic analysis of naphthaleneand anthranilohydroxamic acids, J. Chromatogr., 260 (1983) 493-496. B.A. Bidlingmeyer, Separation of ionic compounds by reversed-phase liquid chromatography: an update of ion-pairing techniques, J. Chromatogr. Sci., 18 (1980) 525-539.