1
1.1
INTRODUCTION
The The met hods hods For For t he det ect i on of t he f unct unct i onal onal
gr oup oup
i n an or gani c compoun ounds i nvol vol ve char char act er i st i c che chemi cal cal r eact i ons.
Dur i ng t he past
avai l abl e tha that
gr oup oups.
Most
t ype ypes of of s pect r oscopy, scopy,
I R spect r oscopy, oscopy, spe sp ect r oscopy. scopy.
are sti l l
1.1.1 1.1. 1 (1) (1 )
of t hem make use of som s ome or e. g.
UV and vi si bl e spect spect r oscopy, scopy,
nucl ear ear magn agnet i c sp s pect r oscopy an and mass
Al l
t hese i nst r ument ent s ar e expen xpensi si ve and r equi r e
con consi der abl e t i me and ski ski l l che chemi cal cal
i nst r ument ent s ar e made
pr ovi de con consi der abl e i nf or mat i on rega regar di ng
many any f unct unct i onal onal ot her
f ew year s,
f or
the thei r
op oper at i on.
Ther ef or e, 1
met hods f or i dent ent i f i cat i on of t he or gani c compoun ounds popul ar .
General Gener al test for amin amines es Tetraphenyl Tetra phenyl borate test
Ami nes t hat hat have a pKa pKa of 11 or l ess may be be det ect ec t ed by t he f or mat i on of wh whi t e pr eci pi t at es wi t h t et r aphenyl bor at e sol sol ut i on.
(2) (2 )
Copper Copp er ion test
2
Wat er sol ubl ubl e l ow mol ecul ar wei ght ami nes nes s uch as ami no al cohol cohol s, sol sol ut i on.
may be det det ect ed by 10 10% w/ v cop copper per
For For mat i on of bl bl ue or bl bl ui sh sh-- gr een col col or at i on or
pr eci pi t at es wi t h t he r eagent ami ne.
sul phat phat e
i ndi cat cat es t he pr esence sence of of an
2
(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, sol ubl e r ed r hodami ne dye i s f or med. some N- ar yl
(4)
ami des i nt er f er
A
Quinhydrone test ’
wat er
Hydr azo compounds and
i n t he t est .
5
Qui nhydr one has been f ound t o be an excel l ent f or t he det ect i on of cer t ai n ami nes.
r eagent
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 ami ne i . e.
pr i mar y,
col or s dependi ng upon t he nat ur e of
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. pr oduce br own col or f ew mi nut es.
t hat
Hinsberg test
Thi s t est
f ade t o yel l ow or col or l ess wi t hi n
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.
(6)
Secondar y ami nes
)
y
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 ony i nal i de to
3
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 4- Ni t r obenzenedi azoni um f l uor obor at e
g
t han ar yl
i s t he most
ami nes.
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
Webst er
9
has pr oposed a ver y si mpl e t est
and secondar y ar yl ami nes. l i gni n i n newspr i nt appl i ed on newspr i nt aci d.
paper . paper
The t est
f or pr i mar y
depends on t he act i on of
Et hanol i c sol ut i on of
ami nes
and t r eat ed wi t h 6N hydr ochl or i c
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 .
4
(9)
Miscellaneous
Primary to
produce 10
b e n z e n e
a nd
secondary
bl ue , test
red
is
or
ary lamines
brown
also
used
of
primary
W a t e r
aliphatic,
soluble
P r i ma r y
isocya nide s o d iu m
test
and
other
S e c o n d ar y A c o n i t i c
f or
aroma tic
Hantzsc h
and
for
is
detected
the
reac tion
with
13 14
amines.
the
detection
a m i ne s .
by
Rimini
test.
’
citric
dia zot ization, glutaconi c
alde hyde
rev eals
Therefor e, th e
wi t h
nic kledithiocarbamate.
acid-acetic
anhydride
1B
that
primary it
was
detec tion
there
is
no
ali phati c desirable of
the
spe cif ic as
to
well fi n d
g on
1
’
, the
acetylace tone-fo rmaldeh yde
pri ma ry
r e a g e nt
to
as out
prim ary
amino
amin es
react
produce
1 f\
tests
compounds.
r e a c t i o n,
12
15
amines.
of
2
, 5-nitr oso-B-h ydroxyq uinolin e ,
detected
and
chloranil
of
heteroc yclic
by
tertia ry
for
employed
detected
detecti on
reagen t
containing
In w i th
th e
17
is
surve y
a mi n e s .
general
group
for
Lit era tu re
reagent
a
by
detection
are
reagents.
a m i ne
anhydride
perf orme d
,
amines , or
is
with
2 , 4 - D m i t r o f l u o r o-
the
alkylamine
pentacyano aquofer rate
furfural
are
2
for
aromatic
primary
a r o ma t i c
c ol o r .
11
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
react
yellow
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
,
5
a qu eo us
s o l ut i o n.
a pp l i c a t i o n of
T he r e f o r e ,
wa s
t h e r e ac t i o n t o d e t e c t
I n t he p r e s e nt
wo r k ,
t he
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 d e t e c t i o n of
it
t h ou gh t
t o e x t e nd t h e
a mi n e s .
r e a c t i o n c o n di t i o ns t o e s t a bl i s h
t h e p r i ma r y a mi n e i n a q u e o u s
a t es t
me d i u m.
of
Ha n t z s c h
f or
t he
6
1. 2
EXPERI MENTAL
2. 3. 2
Appar at us
1.
Corni ng
volum etri c
2.
Corning
3.
Borosil
4.
Sy str oni c
5.
Double
25,50
gradua ted
pipette
2,5
measuri ng
cylinder
pH
beam
m a t c h e d 6.
flask
and
25,50
100ml 10ml
and
capacity.
capacity.
100ml
capacity.
meter. Backman
cell
Constant
and
of
Model-25
1cm
light
tempera ture
spec trophotom eter
having
path.
water
bath
(Townson
and
Mercer
L t d. ,
En gl an d).
1. 2. 2
Reagent
andMat er i al s
Sulphanilanide hydrochloride
B .P . ,
M o n o e t h a n o l a m i n e ( SD 's ) ,
Thiamine
p-Aminobenzoic
p-Toludine
I.P.,
Isomazid
hydrochloride
I .P .,
Sodium
I .P .,
( S D' s ),
I .P .,
hydrochloride
Ethylenediamine
I .P .,
Cyst inehydroc hloride ( SD 's ), ( Su n
Glutamic
Pharma),
I. P. ,
acid
Sodium
acetate
(anhydrous
double
d isti lled
water
U . S .P . ,
Aminophylline sulphate
L-Asparagine ( SD ' s) ,
( B DH ) , used
(BDH),
Nor-Valine acid
Formal deh yde
Acetic
acid
in
st u d y.
the
I .P .,
( M e r ck ) ,
5 -Aminosalicylic
(SD's),
were
L-Lysine
Calcium pentothanete
L-Leucine
Acet yla cet one
I .P .,
( S D' s ),
I . P. ,
I .P .,
a c i d
Glycine
(SD's),
( S D 's ) ,
Procaine
Anthranilic
sulphate
( S D' s ),
Dihyd ralazine
l-Alanin
I .P .,
«<-NaphthyFamine
I.P.,
Streptomycin
Phenylhydrazine
(SD's),
PAS
L-Arginine
hydrochloride
M e t h i o n i n e
B .P . ,
a c id
( S D 's ) ,
( S D' s ) ,
and
7
1.2.2.1
Preparation of standard amine solution
T h e s o l u t i o n a cc ur a t e l y ,
of
we i g h e d
a mi n e s
q ua n t i t y
mi n i mu m q u a n t i t y
of
gi v e t h e d es i r e d
f i nal
1.2.2.2
we r e of
e t h a n ol
pr epar ed
a mi n e
a nd
by d i s s o l v i n g
i n wa t e r
di l u t e d
or
wi t h
c o nc e n t r a t i o n of
wa t e r
t h e a mi n e
Preparation of Acetate Buffer solution
T h e b u f f e r s o di u m a c et a t e s u f f i c i e nt
s ol ut i on of ( 8. 4g)
wa t e r
to
and
p H 4 . 7 wa s
gl aci al
p r o d uc e
ac i d
The
to ( T abl e
by d i s s o l v i n g
( 3 . 3 5 ml )
f i na l
I).
21
pr epar ed
a ce t i c
1 0 0 0 ml .
in a
p H of
in
a b uf 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
di s t i l l e d a c et y l a c et o ne
( 7 . 8 ml )
a c e t a t e b uf f e r
( pH 4. 7) .
Fr eshl y
1.2.3
sol uti on
pr e pa r e d r e ag en t
1 0 0 ml
wi t h
s o l u t i o n wa s u s e d .
Procedure
S t a nd ar d r e ag e nt wa s
and d i l ut e d t o
f r eshl y
sol ut i on
sol uti on
i mme r s e d
cool ed to
of
( 5 . 0 ml )
a mi n e
( 2 . 0 ml )
i n a t es t
i n a b o i l i n g wa t e r
r o o m t e mp e r a t u r e .
t ube.
bat h
t o a 2 5 ml
h el p o f
wa s
T h e a b s o r b a n c e a ga i n s t
t he
T h e v o l u me of
t he c ol or ed
r e a ge nt
b l a nk
f or
2 0 mi n .
s ol ut i on I ).
of
t he
t o ma r k wa s
It
wa s
t h en
t h e t u b e we r e
v o l u me t r i c
adj ust ed
( T a bl ' e
mi x e d wi t h
T h e r e a c t i o n mi x t u r e
T he c o n t e n t s
t r a ns f e r r e d q ua nt i t a t i v e l y wa t e r .
wa s
f l a s k wi t h
t he
wi t h wa t e r .
me a s u r e d a t
415
nm
8
1. 3
RESULTS AND DI SCUSSI ON
T h e p r e s e n c e
of
a mi n o
gr oup
i n t h e c o mp o u n d
has
been
d et e c t e d by v a r i o us
tests.
T he r e
i s no g en er a l
r e ag en t
de t e c t
p r i ma r y
a mi n e s
( a l i p ha t i c ,
a r o ma t i c
al l
t y pe s
he t e r o c yc l i c ) . t o e s t a bl i s h c o mp o u n d s .
of
T he r e f o r e , a tet
f or
Ha nt z s c h
p r i ma r y
T he s o l u t i o n o f
r e a c t i o n wa s
a mi n o
gr o up
a mi n e wa s
( pH 4. 7) , col or
i n b o i l i n g wa t e r
pr oduct
f o r me d wa s
b at h
for
me a s u r e d
c on t a i n i n g
of
a c et a t e
2 0 mi n .
at
„C0CH-
CH„ C0CH, | 2 3
+
I
T we n t y we l l
r e age nt .
as
I ).
^H
H, C- C=0 Q=C- CH, 5 NH, 3 I 2 R
as
buf f e r
4 1 5 n m ( T a bl e
H, CQC
i
t he
The y el l o w
HCHO H, C0CCH„ 3 i 2
and
mo d i f i e d
r e a c t e d wi t h
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
to
s even a mi n o
H 3 C ■^ v
n /
€ H 3
R
al i phat i c ac i d s
The t es t
and t e r t i a r y
x
a mi n e s
h av e
a n d a r o ma t i c be e n d e t e c t e d
i s speci f i c do not
p r i ma r y
f or
by
p r i ma r y
gi v e p os i t i v e
the
a mi n e s .
t e st .
a mi n e s pr o p o s e d S ec o nd ar y
9
TABLE
I
DETECTI ON OF AMI NES Sr . No . Name of
Compo un d
1
S ul pha ni l a mi d e
2
p- Ami nob enz oi c
3
P r o c ai ne
4
p- Tol u di ne
5
q £ -
Conce nt r at i on mol es / l i t x l 0 ~5
aci d
h yd r o c hl o r i de
Na p ht h y l a mme
Absor bance at 415 nm
10. 0
0. 114
2. 5
0. 482
2. 5
0. 184
10. 0
0. 180
2. 5
0. 137
2. 5
0. 187
0. 5
0. 167
5. 0
0. 119
6
Mon o et ha no l
7
S o di u m PAS
8
Ant hr ani l i c
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
0.000
15
Cal ci um pent ot hanet e
10. 0
0. 174
16
Phenyl
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
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
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
6. 8
0. 383
27
5- Ami nos al i c y l i c
1. 3
0. 451
ami ne
aci d
hyd r az i ne- HCl
s ul phat e
hydr oc hl or i de
ac i d ac i d