1.1 INTRODUCTION 1.1 INTRODUCTION
Th
The e mmetet hodhods s FoFor r tt hhe e detdet ectect ii on on of of tt hhe e ff ununctct ii ononal al grgr ououpp ii n n aan n oorr ggaannii c comc comppouounndds s ii nnvovoll ve ve chcharar actact erer ii stst ii c chc cheemmii cacall
rr eeaactct ii oonns. s. DuDurr ii nng g tt hhe e ppaast st ff eeww yyeeaarr s, s, ii nnstst rr uummenentt s s aarr e e mmaaddee a
avvaaii ll aabbll e the thaat t pprr oovvii dde e coconnsisi ddeerr aabbll e ie i nnff oorr mmaatt ii oon regn regaarr ddii nngg m
manany fy f ununctct ii ononal al ggrr ououpps. s. MMost ost oof f tt hhemem mmake ake uuse se of sof somome e oorr o
ott hheer r tt ypypees os of sf s ppeectct rr ooscopscopy, y, ee.. gg. . UUV V aannd d vivi sisi bbll e e spespectct rr ooscopscopy,y, II R R spectspect rr oscoposcopy, y, nnuclucl eaear r mmagagnnetet ii c sc sppectect rr oscopy aoscopy annd d mmaassss
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speectct rr ooscopscopy. y. AAll l l tt hheese se ii nnstst rr uummenentt s s aarr e e eexpxpenensisi ve ve aannd d rr eeqquuii rr ee co
connsisi ddeerr aabbll e e tt ii mme e aannd d skskii ll l l ff oor thr theeii r or oppeerr aatt ii oonn. . TThherer eeff oorr ee,, 11 ch
cheemmii cacal l mmeett hhoodds s ff oor r ii ddenentt ii ff ii catcat ii on on oof f tt hhe e oorr ggaannii c comc comppouounnddss a
are re stisti ll l l ppooppuull aarr ..
1.1.
1.1.1 1 GenerGeneral al test test for for aminamineses (1
(1) ) TetraTetraphenyl phenyl borate borate testtest A
Ammii nes tnes t hahat t have have a a pKpKa a of of 111 1 oor r ll ess ess mmay bay be e detdet ecectt eded b
by y tt hhe e ff oorr mmaatt ii oon n oof wf whhii tt e e pprr eecici ppii tt atat es es wwii tt h h tt eett rr aapphheennylyl b
boorr aatt e soe soll uutt ii oonn..
2 2 (2
(2) ) CoppCopper er ion ion testtest W
Watat er er solsol ububll e le l oow w mmolol eculecul ar ar wweiei gght ht amamii nenes ss s uuch ch aass am
amii nno o alal cohcoholol s, s, mmay ay bbe e dedett ectect ed ed bby 1y 100%% ww// v cov copppeper r sulsul phphatat ee so
soll uutt ii oonn. . FoForr mmaatt ii oon n oof bf bll uue e oor br bll uuii shsh-- ggrr eeeen n cocoll oorr aatt ii oon n oorr p
prr eecici ppii tt aatt ees ws wii tt h h tt hhe e rr eeaaggeennt t ii nnddii cacatt ees ts t hhe e pprr eesensence oce of f aann am
(3) Fluorescein chloride test^
When ami ne sal t i s f used wi t h f l uor escei n chl or i de at 230- 260° i n pr esence of anhydr ous zi nc chl or i de, wat er sol ubl e r ed r hodami ne dye i s f or med. Hydr azo compounds and some N- ar yl ami des i nt er f er i n t he t est .
A 5
(4) Quinhydrone test ’
Qui nhydr one has been f ound t o be an excel l ent r eagent f or t he det ect i on of cer t ai n ami nes. Qui nhydr one r eact s wi t h al i phat i c and ar yl ami ne i n aqueous or i n et hanol i c
medi um t o gi ve di f f er ent col or s dependi ng upon t he nat ur e of ami ne i . e. pr i mar y, secondar y or t er t i ar y ami ne. Ni t r
o-subst i t ut ed ami nes can not be det ect ed by t he r eagent .
(3) N-Halosuccinimide test**
N- Br omosucci ni mi de and N- i odosucci ni mi de ar e good
det ect i ng and di f f er ent i at i ng r eagent s f or ami nes. Pr i mar y and t er t i ar y ami nes r eact wi t h N- i odosucci ni mi de t o gi ve
br own col or t hat does not f ade on heat i ng. Secondar y ami nes pr oduce br own col or t hat f ade t o yel l ow or col or l ess wi t hi n
f ew mi nut es. Ter t i ar y ami nes gi ve an or ange pr eci pi t at es
)
wi t h N- br omosucci ni mi de.
y
(6) Hinsberg test
Thi s t est i s based on t he f act t hat pr i mar y and secondar y ami nes r eact wi t h benzenesui r onyi nal i de to
f or m N- subst i t ut ed sul phonami des, wher eas t er t i ar y ami nes do not r eact wi t h t he r eagent . Fur t her t he sul f onami des of pr i mar y ami nes ar e sol ubl e i n al kal i ne sol ut i on and may be di f f er ent i at ed f r om t he sul f onami de of secondar y ami nes whi ch ar e i nsol ubl e i n al kal i .
(7) Diazonium salt test
Host of t he ar yl ami nes and phenol s under go coupl i ng
r eact i ons wi t h di azoni um sal t s to f or m col or ed azo compounds. Al kyl ami ne gi ves l ess i nt ense yel l ow col or t han ar yl ami nes.
g
4- Ni t r obenzenedi azoni um f l uor obor at e i s t he most sat i sf act or y r eagent out of var i ous di azoni um sal t s empl oyed f or t he t est . I t does not r eact wi t h bot h or t ho and par a posi t i ons subst i t ut ed ami nes.
(8) Lignin test 9
Webst er has pr oposed a ver y si mpl e t est f or pr i mar y and secondar y ar yl ami nes. The t est depends on t he act i on of l i gni n i n newspr i nt paper . Et hanol i c sol ut i on of ami nes
appl i ed on newspr i nt paper and t r eat ed wi t h 6N hydr ochl or i c aci d. Pr i mar y and secondar y ar yl ami ne pr oduce i mmedi at el y yel l ow or or ange col or , whi l e al kyl and al i cyl i c ami nes r equi r e heat i ng of t he sol ut i on f or col or devel opment .
(9) Miscellaneous P r i m a r y a n d s e c o n d a r y a r y l a m i n e s r e a c t w i t h c h l o r a n i l t o p r o d u c e b l u e , r e d o r b r o w n c o l o r . 2 , 4 D m i t r o f l u o r o -1 0 b e n z e n e t e s t i s a l s o u s e d f o r t h e d e t e c t i o n o f a m i n e s . 1 1 p - D i m e t h y l a m i n o b e n z a l d e h y d e i s e m p l o y e d f o r t h e d e t e c t i o n o f p r i m a r y a l i p h a t i c , a r o m a t i c a n d h e t e r o c y c l i c a m i n e s . 2 W a t e r s o l u b l e p r i m a r y a l k y l a m i n e i s d e t e c t e d b y R i m i n i t e s t . P r i m a r y a r o m a t i c a m i n e s a r e d e t e c t e d b y d i a z o t i z a t i o n , 2 1 2 i s o c y a n i d e t e s t , o r b y t h e r e a c t i o n w i t h g l u t a c o n i c a l d e h y d e , 1 3 s o d i u m p e n t a c y a n o a q u o f e r r a t e , 5 - n i t r o s o - B - h y d r o x y q u i n o l i n e , 1 4 1 5 f u r f u r a l a n d o t h e r r e a g e n t s . ’ 1 f\ S e c o n d a r y a m i n e i s d e t e c t e d w i t h n i c k l e d i t h i o c a r b a m a t e . 17 1B A c o n i t i c a n h y d r i d e a n d c i t r i c a c i d - a c e t i c a n h y d r i d e t e s t s a r e p e r f o r m e d f o r t e r t i a r y a m i n e s . , L i t e r a t u r e s u r v e y r e v e a l s t h a t t h e r e i s n o s p e c i f i c r e a g e n t f o r t h e d e t e c t i o n o f p r i m a r y a l i p h a t i c a s w e l l a s a r o m a t i c a m i n e s . T h e r e f o r e , i t w a s d e s i r a b l e t o f i n d o u t a g e n e r a l r e a g e n t f o r t h e d e t e c t i o n o f t h e p r i m a r y a m i n o g r o u p c o n t a i n i n g c o m p o u n d s . 1 g on I n H a n t z s c h r e a c t i o n , ’ , t h e p r i m a r y a m i n e s r e a c t w i t h a c e t y l a c e t o n e - f o r m a l d e h y d e r e a g e n t t o p r o d u c e y e l l o w c ol or ed N- s ubs t i t ut ed- 3, 5- di ac et y1- 1, 4- di hydr ol ut i di ne i n
a q u e o u s s o l u t i o n . T h e r e f o r e , i t wa s t h o u g h t t o e x t e n d t h e a p p l i c a t i o n o f t h e r e a c t i o n t o d e t e c t a mi n e s .
I n t h e p r e s e n t wo r k , t h e r e a c t i o n c o n d i t i o n s o f Ha n t z s c h r e a c t i o n a r e mo d i f i e d s u i t a b l y t o e s t a b l i s h a t e s t f o r t h e d e t e c t i o n o f t h e p r i ma r y a mi n e i n a q u e o u s me d i u m.
1. 2 EXPERI MENTAL 2. 3. 2 Appar at us 1 . C o r n i n g v o l u m e t r i c f l a s k 2 5 , 5 0 a n d 1 0 0 m l c a p a c i t y . 2 . C o r n i n g g r a d u a t e d p i p e t t e 2 , 5 a n d 1 0 m l c a p a c i t y . 3 . B o r o s i l m e a s u r i n g c y l i n d e r 2 5 , 5 0 a n d 1 0 0 m l c a p a c i t y . 4 . S y s t r o n i c p H m e t e r . 5 . D o u b l e b e a m B a c k m a n M o d e l - 2 5 s p e c t r o p h o t o m e t e r h a v i n g m a t c h e d c e l l o f 1 c m l i g h t p a t h . 6 . C o n s t a n t t e m p e r a t u r e w a t e r b a t h ( T o w n s o n a n d M e r c e r L t d . , E n g l a n d ) . 1. 2. 2 Reagent andMat er i al s S u l p h a n i l a n i d e I . P . , p - A m i n o b e n z o i c a c i d I . P . , P r o c a i n e h y d r o c h l o r i d e B . P . , p - T o l u d i n e B . P . , « < - N a p h t h y F a m i n e I . P . , M o n o e t h a n o l a m i n e I . P . , S o d i u m P A S I . P . , A n t h r a n i l i c a c i d ( S D ' s ) , I s o m a z i d I . P . , S t r e p t o m y c i n s u l p h a t e I . P . , L - L y s i n e h y d r o c h l o r i d e ( S D ' s ) , L - A r g i n i n e ( S D ' s ) , G l y c i n e ( S D ' s ) , T h i a m i n e h y d r o c h l o r i d e I . P . , C a l c i u m p e n t o t h a n e t e U . S . P . , P h e n y l h y d r a z i n e h y d r o c h l o r i d e I . P . , A m i n o p h y l l i n e I . P . , E t h y l e n e d i a m i n e I . P . , D i h y d r a l a z i n e s u l p h a t e ( M e r c k ) , M e t h i o n i n e ( S D ' s ) , l - A l a n i n ( S D ' s ) , L - A s p a r a g i n e ( B D H ) , C y s t i n e h y d r o c h l o r i d e I . P . , L - L e u c i n e ( S D ' s ) , N o r - V a l i n e ( S D ' s ) , G l u t a m i c a c i d ( S D ' s ) , 5 - A m i n o s a l i c y l i c a c i d ( S u n P h a r m a ) , A c e t y l a c e t o n e ( S D ' s ) , F o r m a l d e h y d e ( S D ' s ) , S o d i u m a c e t a t e ( a n h y d r o u s ( B D H ) , A c e t i c a c i d ( S D ' s ) , a n d d o u b l e d i s t i l l e d w a t e r w e r e u s e d i n t h e s t u d y .
1.2.2.1 Preparation of standard amine solution T h e s o l u t i o n o f a mi n e s we r e p r e p a r e d b y d i s s o l v i n g a c c u r a t e l y , we i g h e d q u a n t i t y o f a mi n e i n wa t e r o r i n a mi n i mu m q u a n t i t y o f e t h a n o l a nd d i l u t e d wi t h wa t e r t o g i v e t h e d e s i r e d f i n a l c o n c e n t r a t i o n o f t h e a mi n e ( T a b l e I ) . 21
1.2.2.2 Preparation of Acetate Buffer solution
T h e b u f f e r s o l u t i o n o f p H 4 . 7 wa s p r e p a r e d b y d i s s o l v i n g s o d i u m a c e t a t e ( 8 . 4 g ) a n d g l a c i a l a c e t i c a c i d ( 3 . 3 5 ml ) i n
s u f f i c i e n t w a t e r t o p r o d u c e 1 0 0 0 ml . T h e f i n a l p H o f a b u f f e r s o l u t i o n wa s a d j u s t e d o n pH me t e r .
1.2.2.3 Preparation of reagent solution
F o r ma l d e h y d e ( 1 5 ml ; 3 7 S o w / v ) wa s mi x e d wi t h f r e s h l y d i s t i l l e d a c e t y l a c e t o n e ( 7 . 8 ml ) a n d d i l u t e d t o 1 0 0 ml wi t h a c e t a t e b u f f e r s o l u t i o n ( p H 4 . 7 ) . F r e s h l y p r e p a r e d r e a g e n t s o l u t i o n wa s u s e d . 1.2.3 Procedure S t a n d a r d s o l u t i o n o f a mi n e ( 2 . 0 ml ) wa s mi x e d wi t h t h e r e a g e n t s o l u t i o n ( 5 . 0 ml ) i n a t e s t t u b e . T h e r e a c t i o n mi x t u r e wa s i mme r s e d i n a b o i l i n g wa t e r b a t h f o r 2 0 mi n . I t wa s t h e n c o o l e d t o r o o m t e mp e r a t u r e . T he c o n t e n t s o f t h e t u b e we r e t r a n s f e r r e d q u a n t i t a t i v e l y t o a 2 5 ml v o l u me t r i c f l a s k wi t h t h e h e l p o f wa t e r . T h e v o l u me wa s a d j u s t e d t o ma r k wi t h wa t e r . T h e a b s o r b a n c e o f t h e c o l o r e d s o l u t i o n wa s me a s u r e d a t 4 1 5 n m a g a i n s t t h e r e a g e n t b l a n k ( T a bl ' e I ) .
1. 3 RESULTS AND DI SCUSSI ON T h e p r e s e n c e o f a mi n o g r o u p i n t h e c o mp o u n d h a s b e e n d e t e c t e d b y v a r i o u s t e s t s . T h e r e i s n o g e n e r a l r e a g e n t t o d e t e c t a l l t y p e s o f p r i ma r y a mi n e s ( a l i p h a t i c , a r o ma t i c a n d h e t e r o c y c l i c ) . T h e r e f o r e , H a n t z s c h r e a c t i o n wa s mo d i f i e d t o e s t a b l i s h a t e t f o r p r i ma r y a mi n o g r o u p c o n t a i n i n g c o mp o u n d s . T he s o l u t i o n o f a mi n e wa s r e a c t e d wi t h t h e a c e t y l a c e t o n e - f o r ma l d e h y d e i n p r e s e n c e o f a c e t a t e b u f f e r ( p H 4 . 7 ) , i n b o i l i n g wa t e r b a t h f o r 2 0 mi n . T h e y e l l o w c o l o r p r o d u c t f o r me d wa s me a s u r e d a t 4 1 5 n m ( T a b l e I ) . HCHO H, C0CCH„ 3 i 2 CH„ C0CH,| 2 3
i
+
I
H, C- C=0 Q=C- CH, 5 NH, 3 I 2 R ^ H H, CQC „C0CH-H 3 C ■^v x n / € H 3 R T we n t y s e v e n a l i p h a t i c a n d a r o ma t i c p r i ma r y a mi n e s a s we l l a s a mi n o a c i d s h a v e b e e n d e t e c t e d b y t h e p r o p o s e d r e a g e n t . T h e t e s t i s s p e c i f i c f o r p r i ma r y a mi n e s . S e c o n d a r y a n d t e r t i a r y a mi n e s d o n o t g i v e p o s i t i v e t e s t .DETECTI ON OF AMI NES
Sr . No . Name of Compo un d Conce nt r at i on
mol es / l i t x l 0 ~5
Absor bance at 415 nm
1 S ul pha ni l a mi d e 10. 0 0. 114
2 p- Ami nob enz oi c aci d 2. 5 0. 482
3 P r o c ai ne h yd r o c hl o r i de 2. 5 0. 184
4 p- Tol u di ne 10. 0 0. 180
5 q £ - Na p ht h y l a mme 2. 5 0. 137
6 Mon o et ha no l ami ne 2 . 5 0. 187
7 S o di u m PAS 0. 5 0. 167
8 Ant hr ani l i c aci d 5. 0 0. 119
9 I s oni az i d 10. 0 0. 084
10 St r ept omyc i n s ul phat e 10. 0 0. 154
11 L - t ys i ne hydr oc hl or i de 2. 5 0. 403
12 L - Ar gi ni ne 2. 5 0. 228
13 Gl yc i ne 2. 5 0. 155
14 Thi ami ne hy dr oc hl or i de 10. 0 0 . 0 0 0
15 Cal ci um pent ot hanet e 10. 0 0. 174
16 Phenyl hyd r az i ne- HCl 25. 0 0. 181
17 Ami no ph yl l i ne 1 . 0 0. 113 18 Et hy l ene di ami ne 1. 0 0. 123 19 Di hydr al az i ne s ul phat e 10. 0 0. 114 20 Met hi on i ne 2. 5 0. 246 21 L - Al ani n 2. 5 0. 113 22 L - As par a gi ne 5. 0 0. 113 23 Cys t i ne hydr oc hl or i de 5. 0 0. 150 24 L - L e u c me 5. 0 0. 298 25 No r - v al i ne 5. 0 0. 275 26 Gl ut ami c ac i d 6. 8 0. 383 27 5- Ami nos al i c y l i c ac i d 1 . 3 0. 451
