1

1.1 INTRODUCTION

The methods For the detection of the functional group

in an organic compounds involve characteristic chemical

reactions. During the past few years, instruments are made

available that provide considerable information regarding

many functional groups. Most of them make use of some or

other types of spectroscopy, e.g. UV and visible spectroscopy,

IR spectroscopy, nuclear magnetic spectroscopy and mass

spectroscopy. All these instruments are expensive and require

considerable time and skill for their operation. Therefore,1

chemical methods for identification of the organic compounds

are still popular.

1.1.1 General test for amines

(1) Tetraphenyl borate test

Amines that have a pKa of 11 or less may be detected

by the formation of white precipitates with tetraphenyl

borate solution.

2
(2) Copper ion test

Water soluble low molecular weight amines such as

amino alcohols, may be detected by 10% w/v copper sulphate

solution. Formation of blue or bluish-green coloration or

precipitates with the reagent indicates the presence of an

amine.
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(3) Fluorescein chloride test^

When amine salt is fused with fluorescein chloride

at 230-260 in presence of anhydrous zinc chloride, water

soluble red rhodamine dye is formed. Hydrazo compounds and

some N-aryl amides interfer in the test.

A 5
(4) Quinhydrone test

Quinhydrone has been found to be an excellent reagent

for the detection of certain amines. Quinhydrone reacts

with aliphatic and arylamine in aqueous or in ethanolic

medium to give different colors depending upon the nature of

amine i.e. primary, secondary or tertiary amine. Nitro-

substituted amines can not be detected by the reagent.

(3) N-Halosuccinimide test**

N-Bromosuccinimide and N-iodosuccinimide are good

detecting and differentiating reagents for amines. Primary

and tertiary amines react with N-iodosuccinimide to give

brown color that does not fade on heating. Secondary amines

produce brown color that fade to yellow or colorless within

few minutes. Tertiary amines give an orange precipitates

)
with N-bromosuccinimide.

y
(6) Hinsberg test

This test is based on the fact that primary and

secondary amines react with benzenesuironyi nalide to

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form N-substituted sulphonamides, whereas tertiary amines

do not react with the reagent. Further the sulfonamides

of primary amines are soluble in alkaline solution and may

be differentiated from the sulfonamide of secondary amines

which are insoluble in alkali.

(7) Diazonium salt test

Host of the arylamines and phenols undergo coupling

reactions with diazonium salts to form colored azo compounds.

Alkylamine gives less intense yellow color than aryl amines.

g
4-Nitrobenzenediazonium fluoroborate is the most satisfactory

reagent out of various diazonium salts employed for the test.

It does not react with both ortho and para positions substi

tuted amines.

(8) Lignin test

9
Webster has proposed a very simple test for primary

and secondary arylamines. The test depends on the action of

lignin in newsprint paper. Ethanolic solution of amines

applied on newsprint paper and treated with 6N hydrochloric

acid. Primary and secondary arylamine produce immediately

yellow or orange color, while alkyl and alicylic amines

require heating of the solution for color development.

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(9) Miscellaneous

P r i m a r y and s e c o n d a r y a r y l a m i n e s react wit h c h l o r a n i l

to p r o d u c e b l u e , r e d or b r o w n c o l o r . 2,4 - D m i t r o f luoro-
10
benzene t e s t is a l s o u s e d for t h e d e t e c t i o n o f a m i n e s .
11
p-Dimethylaminobenzaldehyde is e m p l o y e d for t h e d e t e c t i o n

of p r i m a r y a l i p h a t i c , aromatic and h e t e r o c y c l i c amines.

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 is d e t e c t e d by 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 are d e t e c t e d by d i a z o t i z a t i o n ,
2 12
isocyanide test , or by 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 ,
13
sodium pentacyanoaquoferrate , 5-nitroso-B-hydroxyquinoline ,
14 15
furfural and other reagents.

1 f\
S e c o n d a r y a m i n e is 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
Aconitic anhydride and citric a c i d - a c e t i c a n h ydride tests

are p e r f o r m e d for 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 is no s p e c i f i c

reagent for 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 as w e l l as

aromatic amines. Therefore, it w a s d e s i r a b l e to f i n d o u t

a g e n e r a l r e a g e n t for 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

group containing compounds.

1 g on
In H a n t z s c h r e a c t i o n , , the pri m a r y amines react
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 to p r o d u c e y e l l o w
colored N-substituted-3,5-diacety1-1,4-dihydrolutidine in
 5

aqueous solution. Therefore, it was thought to extend the

application of the reaction to detect amines.

In the present work, the reaction c on ditions of Hantzsch

reaction are modified suitably to establish a test for the

detection of the primary amine in aqueous medium.

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1.2 EXPERIMENTAL

2.3.2 Apparatus

1. Corning volumetric flask 25,50 and 100ml capacity.

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 and 10ml c a p a c i t y .

3. Borosil m e a s u r i n g c y l i n d e r 25,50 and 100ml capacity.

4. S y s t r o n i c pH meter.

5. Double beam Backman Model-25 spectrophotometer having

m a t c h e d cell of 1cm light path.

6. Constant temperature water bath ( T o w n s o n and M e r c e r Ltd.,

England).

1.2.2 Reagent andMaterials

Sulphanilanide I . P. , p-Aminobenzoic acid I.P., Procaine

h y d r o c h l o r i d e B.P., p-Toludine B.P., <-NaphthyFamine I.P.,

Monoethanolamine 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

(SD's), Isomazid I.P., Streptomycin sulphate I . P ., L-Lysine

hydrochloride (SD's), L-Arginine (SD's), Glycine (SD's),

Thiamine hydrochloride I . P ., C a l c i u m p entothanete U.S.P.,

Phenylhydrazine hydrochloride I .P ., Aminophylline I.P.,

Ethylenediamine I .P ., Dihydralazine sulphate (Merck),

Methionine (SD's), l-Alanin (SD's), L-Asparagine (BDH),

Cystinehydrochloride I . P ., L-Leucine (SD's), Nor-Valine

(SD's), G l u tamic acid (SD's), 5-Aminosalicylic acid

(Sun P h a r m a ) , Acetylacetone (SD's), Formaldehyde (SD's),

Sodium acetate (anhydrous (BDH), Acetic acid (SD's), and

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 in t h e s t u d y .
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1.2.2.1 Preparation of standard amine solution

The s o l u t i o n of a m i n e s w e r e p r e p a r e d by d i s s o l v i n g

accurately, w e i g h e d q u a n t i t y of a m i n e in w a t e r or in a

m i n i m u m q u a n t i t y of e t h a n o l and d i l u t e d w i t h w a t e r to

give the d e s i r e d final c o n c e n t r a t i o n of the a m i n e (Table I).

21
1.2.2.2 Preparation of Acetate Buffer solution

The b u f f e r s o l u t i o n of pH 4.7 was p r e p a r e d by d i s s o l v i n g

s o d iu m a c e t a t e (8.4g) and g l a c i a l a c e t i c aci d (3.35 m l) in

s u f f i c i e n t w a t e r to p r o d u c e 1000ml. The final pH of a b u f fe r

s o l u t i o n was a d j u s t e d on pH meter.

1.2.2.3 Preparation of reagent solution

Formaldehyde (15ml;37So w / v ) w as m i x e d w i t h fre sh l y

distilled acetylacetone ( 7.8ml) and d i l u t e d to 100ml w i t h

acetate buffer solution (pH 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 was used.

1.2.3 Procedure

S t a n d a r d s o l u t i o n of a m i n e ( 2.0ml) was m i x e d wit h the

r ea g e n t s o l u t i o n (5.0ml) in a test tube. The r e a c t i o n m i x t u r e

was i m m e r s e d in a b o i l i n g w a t e r b a t h for 20 min. It was then

c o o le d to roo m t e m p e r a t u r e . The c o n t e n t s of the tub e were

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 to a 25ml v o l u m e t r i c flask w i t h the

help of water. The v o l u m e was a d j u s t e d to mar k w it h water.

The a b s o r b a n c e of the c o l o r e d s o l u t i o n was m e a s u r e d at 415 nm

a g a i n s t the r e a g e n t b l a n k (Tabl'e I).

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1.3 RESULTS AND DISCUSSION

The p r e s e n c e of a m i n o g r o u p in the c o m p o u n d has been

d e t e c t e d by v a r i o u s tests. T he re is no g e n e r a l r e a g e n t to

d e t e c t all types of p r i m a r y a m i n e s ( a l i p h a t i c , a r o m a t i c and

heterocyclic). T he r e f o r e , H a n t z s c h r e a c t i o n was m o d i f i e d

to e s t a b l i s h a tet for 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

compounds. The s o l u t i o n of a m i n e was r e a c t e d w it h the

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 in p r e s e n c e of a c e t a t e b u f fe r

(pH 4.7), in b o i l i n g w a t e r b at h for 20 min. The y e l l o w

c ol or p r o d u c t f o r me d was m e a s u r e d at 415 nm (Ta bl e I).

HCHO ^H
H,CQC C0CH-
H,C0CCH CHC0CH,
3 i 2 | 2 3
i + I
H,C-C=0 Q=C-CH, H 3 C ^v x n / H 3
5 NH, 3
I 2 R
R

T w e n t y s e v e n a l i p h a t i c and a r o m a t i c p r i m a r y a m i n e s

as well as a mi no a c i d s have bee n d e t e c t e d by the p r o p o s e d

rea ge n t. The test is s p e c i f i c for p r i m a r y ami ne s . Secondary

and t e r t i a r y a m i n e s do not g iv e p o s i t i v e test.

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TABLE I

DETECTION OF AMINES
S r .N o . Name of C om po und Concentration A b s o r ba nc e
moles/litxl0~5 at 415 nm

1 S ul ph a ni la m id e 10.0 0.114
2 p - A m i n o be n zo i c acid 2.5 0.482
3 P ro caine h y d r o c hl or id e 2.5 0.184
4 p-Tolu dine 10.0 0.180
5 q - Naphthylamme 2. 5 0.137
6 M o no et han ol amine 2 .5 0.187
7 Sodium PAS 0.5 0.167
8 A nt hr a ni li c acid 5.0 0.119
9 Isoni az id 10.0 0.084
10 S tr ep t om y ci n s ul phate 10.0 0.154
11 L -t ys ine h y dr o c h l o r i d e 2.5 0.403
12 L-Argi ni ne 2.5 0.228
13 Glycine 2.5 0.155
14 Thiamine h y d r o c hl or id e 10.0 0.000
15 Calcium p e nt o tha ne te 10.0 0.174
16 Phenyl h yd r az i ne - HC l 25.0 0.181
17 Amin oph yl lin e 1 .0 0.113
18 E th yl e ne d ia mi n e 1.0 0.123
19 Dihydralazine s ulphate 10.0 0.114
20 M et hi on i ne 2.5 0.246
21 L-Alanin 2.5 0.113
22 L -A sp ara gi ne 5.0 0.113
23 Cystine h yd r oc h lo ri de 5.0 0.150
24 L-Leucme 5.0 0.298
25 Nor-va li ne 5.0 0.275
26 Glutamic acid 6.8 0.383
27 5 - Am i no s a l i c y l i c acid 1 .3 0.451