31
CHAPTER - II
DETERMINATION OF NITRITES
2.1
Introduction :
Inorganic and organic nitrites (nitrates) have a common
property of relaxing smooth muscles including that of blood
vessels.
In moderate doses, they have little effect on blood
pressure but can produce marked postural hypotension and
synocope due to the
relaxations of veins.
101
Nitrites and
nitrates are common constituents of many food stuffs,
either occuring naturally or used as food additives.
102
The
role of nitrite as a precursor in the formation of carcino­
genic N-nitrosamines has generated an
increased interest in
the analysis of this compound in foods and other sources.
Various methods suggested for the determination of
nitrites are summarized below :
2.2
Methods for nitrite determination :
2.2.1 Titrimetry :
(a)
Acidimetry :
Nitrites are titrated directly with perchloric acid in
ethyleneglycol-propanol
(or chloroform) as well as propylene-
glycol-propanol (or chloroform) mixture.
The end point of
the titration is detected by using internal indicator or
103
potentiometrically.
Nitrites are titrated potentiometri-
eally with pyridine-perchloric acid in DMF-water (1:1) mixture
32
as medium.
104
Nitrites are also titrated potentiometrically
with tetrabutylammonium hydroxide solution in benzene-methanol
medium. 105
Ethylnitrite is formed in the reaction of sodium
nitrite and ethanol in presence of sulphuric acid.
The excess
of sulphuric is determined by titration against standard
sodium hydroxide solution using phenolphthalein
as indicator.
106
Sodium nitrite has been determined in 30% v/v ethanol
medium by high frequency titration with 0.1 N hydrochloric
acid.
107
The end point was measured at 130NHZ
in 130ml total
volume and corresponded to the formation of un-ionized nitrous
acid.
The determination of 10-51 mg of sodium nitrite was
unaffected in the presence of 50-200mg of potassium nitrate
and 50~100mg of ammonium chloride.
(b)
Oxidimetry :
The usual oxidation reduction titration method for
determination of nitrite is reported in which nitrite is
oxidized to nitrate with strong oxidizing agent.
It is also
possible to reduce nitrite to a variety of products, depending
upon the nature of the reducing agent.
Details of redox
procedures are discussed below :
(i)
Hypochlorite :
Nitrites are directly titrated with hypochlorite solution
103
potentiometri*cally or amperometrically.
Alternatively, they
33
are t r e a t e d w i t h h y p o c h l o r i t e s o l u t i o n in p r e s e n c e of s o d i u m
bicarbonate,
the e x c e s s of h y p o c h l o r i t e is th en d e t e r m i n e d
lodometncally.
107
(ii) Chloramine-T :
N i t r i t e s are r e a c t e d w i t h c h l o r a m i n e - T s o l u t i o n in
p r e s e n c e of s o d i u m b i c a r b o n a t e or a c e t i c acid.
of c h l o r a m i n e - T is d e t e r m i n e d i o d o m e t r i c a l l y .
108
The e x c e s s
’
109
They
are al so e s t i m a t e d by d i r e c t t i t r a t i o n w i t h c h l o r a m i n e - T
solution p o te n t i o metrically.
109
N , N - D i m e t h y l f o r m a m i d e as
t i t r a t i o n m e d i u m g i v e s b e t t e r r e s u l t s in the b a c k t i t r a t i o n
.
procedure.
110
(iii) Chlorate :
A p r o c e d u r e b a s e d on o x i d a t i o n of n i t r i t e s w i t h c h l o r a t e
is r e p o r t e d .
The c h l o r i d e f o r m e d is r e a c t e d w i t h s i l v e r
nitrate solution.
The e x c e s s of s i l v e r n i t r a t e is d e t e r m i n e d
by V o l h a r d m e th o d .
(iv)
Bromate :
N i t r i t e s are r e a c t e d w i t h b r o m a t e s o l u t i o n in p r e s e n c e
of h y d r o c h l o r i c acid.
iodometrically.
112
The e x c e s s of b r o m a t e is d e t e r m i n e d
Alternatively,
b r o m i n e in p r e s e n c e of p y r i d i n e .
determined iodometrically.
103
n i t r i t e s are r e a c t e d w i th
The e x c e s s of b r o m i n e is
34
(v)
Cerdc
sulphate :
Nitrite is reacted with standard ceric
solution.
The excess
of ceric
sulphate
sulphate is titrated with
standard sodium oxalate solution using nitroferroin as
indicator.
113 114
’
or is titrated with standard ferrous
ammonium sulfate using ferroin as indicator. 115
(vi) Iodometry :
Nitrite solution is added to the mixture of sodium
bicarbonate,, potassium iodide and amyl alcohol
with acetic acid.
and acidified
Excess of sulphuric acid (1:1) is added
and the liberated iodine is titrated with standard sodium
thiosulphate solution using starch mucilage as indicator. 116
(vii)
Iodimetry :
Nitrites are directly titrated with standard iodine
solution in presence of chloride and nitrogenous material. 116
(viii)
Permangenate :
The nitrite is added to permanganate solution.
The
reaction mixture is acidified and excess of permanganate is
117
determined iodometrically.
not interferin the titration.
The presence of ammonia does
118
35
(ix)
Ferricyanide :
N i t r i t e s are r e a c t e d w i t h f e r r i c y a n i d e .
formed is t i t r a t e d a g a i n s t s t a n d a r d c e r i c
using
(x)
f e r r o i n as i n di c a t o r .
The f e r r o c y a n i d e
sulphate solution
11 y
Sulphonamide :
S u l p h a n i l a m i d e is t i t r a t e d d i r e c t l y w i t h s o d i u m n i t r i t e
u s i n g s t a r c h i o d i d e as i n d i c a t o r . ^ 0
(c)
Reductimetry :
R e a c t i o n of n i t r i t e is not
procedure,
however,
c o m m o n l y us ed as an a n a l y t i c a l
t h e r e are t h r e e r e d u c t a n t s that h a v e be en
e m p l o y e d for q u a n t i t a t i v e d e t e r m i n a t i o n of n i t r i t e m
(i)
solution.
Titanous solution :
N i t r i t e is r e a c t e d w i t h t i t a n o u s c h l o r i d e .
The e x c e s s
of t i t a n o u s c h l o r i d e is t i t r a t e d w i t h s t a n d a r d f e r r i c s o l u t i o n
u s i n g t h i o c y a n a t e as an i n d i c a t o r .
119
(ii) Hydrazine :
N i t r i t e is r e a c t e d w i t h e x c e s s of h y d r a z i n e in p r e s e n c e
of s u l p h u r i c acid.
The e x c e s s of h y d r a z i n e is d e t e r m i n e by
titration with standard iodine solution.
121
Improved results
are r e p o r t e d in d i r e c t p o t e n t i o m e t r i c t i t r a t i o n of h y d r a z i n e
wi th the n i t r i t e s o l u t i o n .
122
36
(iii) Hydroxylamine :
Nitrite solution is mixed with standard hydroxylamine
hydrochloride solution.
The excess of hydroxylamine hydro­
chloride is titrated with standard sodium hydroxide solution
using ethanolic solution of methylene blue and phenol red or
mixed indicator.
2.2.2
UV
123
Spectrophotometric methods :
Nitrite is measured spectrophotometncally at 355nm.
The Lambert-Beer's law is obeyed in the concentration range
of 0.6 - 1.2g of nitrite per liter.
Sodium nitrite is
determined spectrophotometrically in pharmaceutical prepara­
tions by measuring absorbance at 355nm.^^
(i)
Diazonium salt formation :
The diazonium salts formed from sulphanilic acid have
an extremely sensitive ultraviolet absorption spectrum which
has been used successfully for the determination of nitrite.
The maximum absorbance is observed at 270nm.
127
The Lambert-
Beer's law is obeyed in the concentration range of 0.2 - 3.2mg
of nitrite per liter.
Similarly, diazonium salts formed for
phenylenediamine and its derivates have been used for nitrite
determination.
The sensitivity of these diazonium salts is
three to four times greater than that of salts prepared from
sulphanilic acid.
These diazonium salts absorb strongly in
the range of 320-400nm.
1’ 6
37
(ii) Reaction with 2,6-xylenol :
Nitrite is reacted with 2,6-x y le n ol in sul ph u ri c acidwat er - ac e ti c acid (5:4:1) med iu m to form 4 - n i t r o s o - 2 ,6 - x y l e n o l .
The maximum abs or b an c e of reaction m ixture is observed at
307nm.
The Lambert Beer's law is obeyed in the c o n c e n tr a ti on
range of 0-10 ppm of nitrite.
1 27
(iii) Reaction with thioglycolic acid :
Nitrite reacts with t h i o g l yc o li c acid in weakly acid
medium to form S - n i t r o s o t h i o g l y c o l i c acid, having max im u m
abs or b an c e at 355nm.
The L a m b e r t - B e e r 's law is obeyed in
the c o n c e n t r a t i o n range of Q-100mg of nitrite per liter. 12 8
2.2.3 Visible spectrophotometric methods :
The most common p ro ce d ur e s used for the d e t e r m i n a t i o n
of nitrite are based on the formation of an azo dye. Nitrite
is reacted with primary aromatic amine in an acidic solution
to form d i a zo n iu m salt.
The d ia zonium salt when coupled
i
with an aromatic compound having primary amino or phenolic
functional group forms a c on ju g at e d azo dye which is suitable
for col or i me t ri c measurements.
(i)
Sulphanilic acid method :
Dia zo n iu m salt formed by reaction of nitrite with
sulphanilic acid
when coupled with v arious primary aromatic
amines gives azo dye.
The most common coupling agent employed
in this reaction is 1 - n a p h t h y l a m i n e . 129
38
. (ii) Sulphanilamide method :
S u 1 p h a n i 1 a m 1 de is d i a z o t i z e d w i t h n i t r i t e .
The d i a z o n i u m
salt f o r me d is t h a n c o u p l e d w it h N - (1 - n a p h t h y 1) e t h y l e n e d a m i n e
d i h y d r o c h l o r i d e to form azo dye h a v i n g m a x i m u m a b s o r b a n c e at
530nm.
The m e t h o d is r e p o r t e d to be e x t r e m e l y s e n s i t i v e
nitrite d et er m in a ti on . 130-132
for
The L a m b e r t B e e r ' s law is o b e y e d
in the c o n c e n t r a t i o n r a n g e of O.D - 0 . 4 m g of n i t r i t e per liter.
(iii) Reaction with brucine :
A r a p i d c o l o r i m e t r i c m e t h o d b a s e d on the r e a c t i o n b e t w e e n
n i t r i t e and b r u c i n e in s u l p h u r i c aci d m e d i u m to form c h r o m o p h o r e
is r e p o r t e d .133
(iv) Free radical chromogen :
1-Methyl-2-quinolone a z i n e w h e n r e a c t e d w i t h n i t r i t e
g i v e s c o n s i d e r a b l y e n h a n c e d s e n s i t i v i t y at 520nm.
(v)
1 34
Reaction with Thiourea :
N i t r o u s a c i d r e a c t s w i t h t h i o u r e a to form t h i o c y a n i c
a cid w h i c h f orms c o m p l e x w i t h F e 3
at 4 5 5 n m .
ion h a v i n g m a x i m u m a b s o r b a n c e
The m e t h o d is a p p l i e d to the d e t e r m i n a t i o n of 2 - 1 2 p p m
of_ n i.,t r i t e . 135
(vi) Phenylazoaniline method :
N i t r i t e is r e a c t e d w i t h p h e n y l a z o a n i 1 ine in a l k a l i n e
d i m e t h y l f o r m a m i d e to g i v e a t r i a z e n e a n i o n h a v i n g i n t e n s e
blue color.
The m e t h o d has a s e n s i t i v i t y of 0 . 1 5 m c g of
n i t r i t e per m l .
39
(vii) Reaction with rivanol :
Nitrite reacts with rivanol (2-ethoxy-6,9-diaminoacridine
lactate) in 1.8M HC1 to give intense colored product having
maximum absorbance at 515nm.
The Lambert-Beer's law is obeyed
in the concentration range of 0.0 - 1.2mcg per ml.
137
(viii) Reaction with ferrous sulphate :
Nitrite is reacted with Fe^+ ion in 2.5M phosphoric acid.
The ferrous nitrosyl complex ion formed absorbs at 455-46Qnm
and is sensitive to nitrite concentration of 2.5mcg/ml.
presence of nitrates and sulphates do not interfer
determination of nitrite.
(ix)
The
in the
138
Reaction with dimethylaniline :
Nitrous acid reacts with dimethylaniline to form yellow
colored p-nitroso dimethylaniline which is measured colori139
metrically.
2.2.4 Miscellaneous methods :
(i)
Coulometry :
A direct determination of nitrite has been accomplished
by using coulometrically generated manganese (III) as
titrant.140’141
(ii) Polarography :
Several polarographic methods for the determination of
nitrite have been developed.
Nitrite ion gives the same effect
40
as nitrate ion in the reduction.
In a detailed e x a mi n at i on
of reduction of nitrite and nitrate in presence of uranyl ion
showed that the d iffusion current con st a nt s are different.
142
A qua nt i ta t iv e d et er m i n a t i o n of both nitrite and nitrate could
be carried out by d e t e r m in a ti on of the total d if fusion current,
followed by o xidation of nitrite to nitrate with hydrogen
peroxide in acid solution and m e a su r em e nt of the increased
d iffusion current.
(iii)
Amperometric titration :
Nitrites are titrated with Ce 4+ ion in 0.05M sulfuric
acid medium after addition of three drops of 0.01M osmium
tetroxide as catalyst, using a rotating p la ti n um electrode
at a cell p otential of 1.05 volts versus the s at urated calmoel
electrode.
143
The p er ma n g a n a t e a mp er o me t ri c t it ration of
nitrite is c arried out by e mp loying p r e p o l a r i z e d rotating
platinum ele ct r od e s of 1.10 volts versus the s a t ur a te d c alomel
electrodes.
Similarly, sulfamic acid t it ration has been
used suc ce s sf u ll y with rot at i ng platinum ele ct r od e s at 1.05
. ,n
.. 141.144
or 1.10 volts.
41
Studies on Griess reaction
:
It is known that organic nitrites are hydrolyzed to
give nitrite or nitrate ion.
evident
From the above review, it is
that the method based on Griess reaction
145
is sample
and rapid for the determination of micro-quantities of nitrite
in aqueous medium.
It was, therefore, thought of interest to
investigate the reaction in order to increase the sensitivity
of the test.
Griess
was first to describe the formation of pigments
from various nitro compounds (aniline derivatives), nitrous
acid and various coupling agents (naphthalene derivatives).
The
145
reaction is known as Griess reaction and is widely
employed in the analysis of nitrite.
The original Griess reaction
1 47
146
has received considerable
attention and many improvements in the procedure are suggested.
1 29
The reaction takes place in three steps : (i) nitrosation,
(ii) formation of diazonium salt, and (iii) coupling.
The first
step is an acid catalyzed reaction, the second, an internal
rearrangement and the third, a reaction that proceeds at different
rate depending on pH.
For the estimation of nitrite, the
reaction is carried out at a pH that is a compromise between
the optimal pH values for the two pH dependent reactions.
42
Rider and Mellow
and coupling
1 29
reaction.
have examined the diazotisation
They observed that for a success
of nitrite estimation, (a) the diazotisation should be
carried out in a strong acid solution
cool a solution as possible;
and conducted in as
(b) the coupling should be
carried out only after the diazotisation is completed in as
low an acidity as is consistent with colorimetric stability.
However, IlosVay
148
recommends the use of acetic acid which
results in reaction solution, pH values in the range of
maximal conversion for sulfanilic acid/1 -naphthylamine
reaction, but whether this is a universal, is not well
established.^ ^
Sawicki et al
150
investigated the reaction for colori­
metric determination of nitrite, however, no direct comp&Tision
of the relative effectiveness of various Griess reagent combi­
nations has been made.
Fox
1 49
in his studies on Griess reaction observed that
the amount of diazo pigment
formed from the reaction of a
variety of aniline and naphthylamine derivatives is dependent
on (i) kind and concentration of reagent;
(ii) specific
combinations and relative concentrations of nitrosable compounds
and coupling agents; (iii) reaction of nitrite with ring
substituents (other than the amino group) and/or the coupling
43
reagent,
(iv) formation of more than one pigment,
(v) o x i d a ­
tion of the d i a zo n iu m ion int er m ed i at e and/or the pigments,
(vi) oxides of nitro g en in air,
(vii) red uc t io n of the d iazonium
ion by residual reductants (such as a scorbate
ion),
(viii)
formation of s em i-stable n i t r o s o - r e d u c t a n t intermediates, and
(ix) pre -r e ac t io n of nit ro s ab l e compound and nitrite.
addition to pH and t em perature of the reaction,
In
the above
m entioned factors are important.
Lunge et al 1 5 1 and others 1 5 2 have reported the nit ro s ab l e
compound must be in at least 100 fold molar c o n c e n t r a t i o n excess
over that of nitrite for maximal pigment product.
Incomplete
color dev el o pm e nt occurs, if nitrite reacts with coupl i ng agent,
1- n a p h t h y l a n i n e , which is the basis for a method of nitrite
determination.
153
It was, therefore,
thought of interest to
carry out the reaction using various n it ro s ab l e c o m po u nd s and
coupling agents under the standard set of
c on di t io n s and then
picked up the reagent c o m bi n at i on for further study
under
special conditions.
In the present work, various c o m bi n at i on of nit ro s ab l e
compounds and coupling reagents were reacted with n itrite under
standard set of reaction conditions.
Procaine as n it ro s ab l e
compound and N - 1 - ( n a p h t h y l )e th yl e n e d i a m i n e as coupling reagent
are found to give max im u m color intensity which is more than
44
twice that obtained with sulfanilic acid (nitrosable compound)
and 1-naphthylamine (as coupling agent)
(Table-1).
Various
reaction conditions using procaine/N-1(naphthy1)ethylenediamine
combination were standardized to obtain maximum color intensity
of the test.
2.3 Experimental :
2.3.1
1.
Apparatus :
Double beam Hitachi Spectrophotometer Model 210 having
two matched cells with 1 cm light path.
2.
Constant temperature water bath (Townson and Mercer Ltd.,
England).
3.
Corning volumetric flasks of 25,100 and 500 ml capacity.
2.3.2
Reagents and Materials :
Sodium Nitrite (BDH); Procaine Hydrochloride
(BP);
Hydrochloric acid -(BP); N-1(Naphthy1 )ethylenediamine
dihydrochloride (GR); Potassium hydroxide (Pellets (BDH);
Sodium Nitrate (BDH); Anthralinic acid (GR); p-Aminosalicylie
acid (USP); Benzocaine (BP); p-Chloroaniline (GR); o-Nitroaniline (GR);
Sulfanilic acid (BDH); m-Aminophenol
(BDH); sulfamethoxazole (BP);
45
Sulfa n il a mi de (USP); M e t o c l o p r a m i d e h yd ro c h l o r i d e
(BP);
p-A mi n ob e nz oi c acid (USP); 1- N a ph t hy l am in e (BDH);
2 -N ap h th y la mi n e (BDH); 1-Naphthol (BDH); 2-Naphthol (BDH);
Sulphuric acid (GR); Glacial acetic acid (BP); Ethanol (BP);
Sodium h yd roxide (P e l l e t s - B D H ) and double d is tilled water
were used in the study.
Anthralinic acid solution (0.5% w/v) :
A n t hr a li n ic acid (500mg) was d issolved in sodium hydroxide
(10ml,1N) and diluted to 100ml with water.
%
p-Aminosalicylic acid solution (0.5% w/v) :
p -A m i n o s a l i c y l i c acid (500mg) was d i s so l ve d in sodium
hydroxide (10ml,1N) and d iluted to 100ml with water.
Benzocaine hydrochloride solution (0.5% w/v) :
Ben zo c ai n e h yd ro c h l o r i d e (500mg) was d issolved in and
diluted to 100ml with water.
46
p-Chloroani1ine solution (0.535 w/v) :
p-Chloroaniline (500mg) was dissolved in hydrochloric
acid (10ml; 4N) and diluted to 100ml with water.
o-Nitroaniline solution (0.535 w/v) :
o-Nitroaniline (500mg) was dissolved in hydrochloric
acid (10ml; 4N) and diluted to 100ml with water.
Procaine hydrochloride solution (0.5% w/v) :
Procaine hydrochloride (500mg) was dissolved in and
diluted to
100ml with distilled water.
Sulfanilic acid solution (0.5% w/v) :
Sulfanilic acid (500mg) was dissolved in and
diluted
to 100ml with distilled water.
m-Aminophenol solution (0 * 5 % w/v) :
.m-Aminophenol (500mg) was dissolved in sodium hydroxide
solution (10ml; 1N) and diluted to 100ml with water.
Sulfamethoxazole solution (0.5% w/v) :
Sulfamethoxazole (500mg) was dissolved in hydrochloric
acid (10ml; 4N) and diluted to 100ml with water.
Metoclopramide hydrochloride solution (0.5% w/v) :
Metoclopramide hydrochloride (500mg) was dissolved in
and diluted to 100ml with water.
47
p-Aminobenzoic acid solution (0.5% w/v) :
p-Aminob'enzoic acid (500mg) was d issolved in sodium
hydroxide solution (10ml; 1N) and diluted to 100ml with
water.
2-Napthylamine solution (0.1" w/v) :
2 -N ap h t h y l a m i n e (100mg) was dis so l ve d in ethanol (5ml).
Hyd ro c hl o ri c acid (2ml; 4N) was mixed with it and diluted
to 100ml with water.
1- Naphthol solution (0.1% w/v) :
1-
Naphthol (100mg) was dis so l ve d in sodium h ydroxide
(2ml; 1N) with warming and diluted to 100ml with water.
2- Naphthol solution (0.1% w/v) :
2-
Naphthol (100mg) was d issolved in sodium h ydroxide
(2ml; 1N) with slight w arming and diluted to 100ml. with
water.
N-1- (Naphthyl)ethylenediamine dihydrochloride solution
(0.1% w/v) :
N-1-( N ap h th yl )ethylen e di am i ne d i h y d r o c h l o n d e
(100mg)
was dissolved in and d iluted to 100ml with water.
Hydrochloric acid (4NQ :
Concentrated hydrochloric acid (38ml) was diluted to
100ml with water, and standardized against sodium carbonate
anhydrous.
48
Sulfuric acid (4N) :
Concentrated sulfuric acid (12ml) was mixed slowly
with ice cooled water (70ml).
After cooling to room
temperature, the volume was adjusted to 100ml with water
and standardized against anhydrous sodium carbonate.
Acetic acid (4N) :
Glacial acetic acid (24ml) was diluted to 100ml with
water and standardized by titrating against 4N sodium
hydroxide solution.
Sodium nitrate solution (0.15K w/v) :
Sodium nitrate (100mg) was weighed and dissolved in
and diluted to 100ml with water.
Standard sodium nitrite solution :
Sodium nitrite (62.5mg) previously dried at 100°C for
3 hours was dissolved in and diluted to 500ml with water.
An aliquot (5ml) was diluted further to 100ml with the same
solvent.
Final solution contained 6.25mcg of sodium nitrite
per ml of the solution.
2.3.3 Procedures :
2.3.3.1 Determination of wave length of maximum absorbance :
Standard sodium nitrite solution (2.0ml) was pipetted
into a 25ml volumetric flask and diluted to 10ml with water.
Procaine hydrochloric solution (1.0ml; 0.5?^ w/v) was added
49
to it and mixed thoroughly.
The reaction mixture was
allowed to stand for 3- minutes at room temperature.
Hydro­
chloric acid (1ml; 4N) was added to it and mixed.
N-1-(Naphthyl)ethylenediamine dihydrochloride solution
(1.0ml; 0.1 % w/v) was added to it and allowed to stand at
room temperature for 10 minutes.
The volume was adjusted
to 25ml with water and allowed to stand for 30 minutes.
The absorbance of
the colored solution was scanned in the
range of 350 to 650nm against blank.
The blank was prepared similarly in which volume of
standard sodium nitrite solution
volume of water.
was replaced by an equal
Maximum absorbance was obtained at 545nm
(Fig .1 ) .
2.3.3.2 Lambert-Beer's curve for sodium nitrite solution :
Standard sodium nitrite solution (0.2, 0.4, 0.8, 1.2,
1.6, 2.0, 2.5 and 3.0ml) was pipetted into a series of 25ml
volumetric flasks and was treated as described under 2.3.3.1
The absorbance of the colored solution was measured at 545nm
against reagent blank (Fig.2).
2.3.4
Factors affecting the development of color :
2.3.4.1 Effect of diazotising
agents and coupling agents :
Standard sodium nitrite solution (2.0ml) was pipetted
into a series of 25ml volumetric
flasks and diluted to 10ml
50
with water.
The solutions of different aromatic primary
amines(Table I) was added to it and mixed throughly.
The
reaction mixture was allowed to stand for 3 minutes.
Hydrochloric acid (1ml, 4N) was added to each reaction
mixture and mixed with various coupling reagent solutions
(1.0ml) (Table I).
The reaction mixture was allowed to stand
at room temperature for 10 minutes.
to 25ml with water and allowed to
room temperature.
The volume was adjusted
stand for 30 minutes at
The absorbance of the colored solutions
was measured at 545nm against respective reagent blank
(Table I).
2.3.4.2 Effect of concentration of procaine hydrochloride
solution :
Standard sodium nitrite solution (2.0ml) was pipetted
into a series of 25ml volumetric flasks and diluted to 10ml
with water.
Different volumes of procaine hydrochloride
solution were added to it and treated as described under
2.3.3.1. The absorbance was measured at 545nm against blank.
The maximum absorbance was obtained in presence of
1ml of procain<Lhydrochloride solution which remained constant
on increasing the concentration of procaine hydrochloride
solution (Fig.3).
51
2.3.4.3
Effect of various acids on diozatisation :
Standard sodium nitrite solution (2.0ml) was pipetted
into a series of 25ml volumetric flasks and diluted to 10ml
with water.
Procaine hydrochloride (1.0ml, 0.5.w/v) was
added and mixed throughly.
The reaction mixture was allowed
to stand for 3 minutes at room temperature. . The hydrochloric,
sulfuric
or acetic acid (1ml, 4N) was added to it and
treated as described under 2.3.3.1.The absorbance of the
colored solutions were measured at 545nm against the respective
reagent
blank (Table II).
Maximum absorbance was obtained in presence of
hydrochloric acid (Table II).
2.3.4.4 Effect of concentration of hydrochloride acid :
Standard sodium nitrite solution (2.0ml) was pipetted
into a series of 25ml volumetric flasks and diluted to 10ml
with water.
Procaine hydrochloride solution (1ml; 0.5% w/v)
was added to it and mixed with different volumes of hydro­
chloric acid (4N) and treated as described 2.3.3.1. The
absorbance was measured at 545nm against the reagent blank.
The maximum absorbance was obtained in presence of
1ml of hydrochloric acid (4N) which decreased slightly on
increasing the concentration of hydrochloric acid in the
reaction mixture (Fig.4).
52
2-3. 4. 5 m e e t of concentration oT N-1- (naphthyl)ethyIene,'Uiuinine hydrochloride reuyeol uoluLion :
Standard sodium nitrite solution (2.0ml) was pipetted
into a series of 25ml volumetric flasks and diluted to 10ml
with water and treated as described under 2.3.3.1 using
different volumes of the reagent solution (0.1% w/v).
The
absorbance was measured at 545nm against the reagent blank.
The maximum absorbance was obtained in presence of
1.0ml of reagent solution which remained constant on
increasing the reagent concentration (Fig.5).
2.3.4.6
Effect of temperature :
The reaction of standard sodium nitrite solution (2.0ml)
with procaine hydrochloride was carried at different temperature
(3
,10°,22°,30°,37°,55°,75° ) and treated as described under
2.3.3.1.
The absorbance of colored solution was measured at
545nm against the reagent blank.
The color'intensity of the reaction mixture between
30° to 60°, however, it decreases with the further increase
in temperature (Fig.6).
2.3.4.7
Effect of time :
Standard sodium nitrite solution (2.0ml) was treated
as described under 2.3.3.1 and allowed to stand for different
time intervals (5,10,15,20,120 min) after dilution.
The
53
absorbance of colored product was measured at 545nm against
the reagent blank (Fig.7).
Maximum color intensity was obtained after 20 min
which remained constant upto 2 hours.
2.3.4.8 Interference of nitrate ions :
Standard sodium nitrite solution (2.0ml) was pipetted
into a series of 25 ml volumetric flasks.
of sodium nitrate solution (1.0mg/ml)
Different volumes
(1.0,3.0,4.0 and 6.0ml)
was added to each flask and diluted to 10ml with water and
treated as described under 2.3.3 .1 . The absorbance was measured
at 545nm against the reagent blank.
No interference of nitrate ion was observed (Fig.8).
54
2 .4
RESULTS AND DISCUSSION
In the preliminery experiments, anthralinic acid,
p-aminobenzoic acid, benzocaine, procaine, p-aminosalicylic
acid, o-Nitroaniline, p-chloroaniline, m~aminophenol, sulfanilic
ncid, r.u1phnn i 1nmi de , nul fmnothoxnznl r , nnd inntno 1np nun i(In worn
used as diazotising amines, while 1-naphthylsmine, 2-naphthylamine, N-1-(naphthyl)ethylenediamine, 1-naphthol and 2-naphthol
were employed as coupling reagents. The reaction of nitrite
with nitrosable compound and coupling agent combination was
carried out in dilute hydrochloric acid medium
set of reaction conditions (Table-I).
under standard
The reaction mixture
containing procaine/N-1-(naphthyl)etbylenediamine gives the maximum
color intensity.
Out of the three
acids, hydrochloric acid
is found to be most satisfactory (Table-II),
In the proposed procedure, the reaction mixture containing
nitrite and procaine hydrochloride was acidified with hydro­
chloric acid (pH 0.58).
The diazonium salt.formed is then
coupled with N-1-(naphthyl)ethylenedaimine to give purple
colored product having .maximum absorbance at 545nm (Fig.1).
The optimization of various reaction conditions revealsthat
maximum color intensity is obtained when (i) the molar concen­
tration of procaine and N-1-(naphthyl)ethylenediamine were 150
and
31 fold of nitrite concentration respectively (Fig.3
(ii) the diazonium reaction was carried out at
0.58 pH.
&
5),
55
at 30° ( F i g . 4),
(iii) c o u p l i n g r e a c t i o n w as c a r r i e d out a f t e r
the d i a z o t i z a t i o n was c o m p l e t e
(Fig.
), (iv) c o u p l i n g w i t h
N - 1 - ( n a p h t h y l )e t h y l e n e d i a m i n e w a s c a r r i e d out at pH 0 . 85
20 min.
( F i g . 7).
concentration
The L a r n b e r t - B e e r 's law is o b e y e d
for
in the
r a n g e of 0. 0 to U.7 meg of n i t r i t e pur ml ol
the r e a c t i o n m i xt u r e .
The proposed
sodium nitrite.
p r o c e d u r e was a p p l i e d to the d e t e r m i n a t i o n
The r e s u l t s c o m p a r e d f a v o r a b l e
of s u l f a n i l i e / 1 - n S p h t h y l a m i n e
m e th o d ,
however,
w i t h t h os e
the
s e n s i t i v i t y of the p r o p o s e d m e t h o d is a b o u t t w ic e that of reported
m e th o d .
The p r e s e n c e of n i t r a t e ion d o e s not i n t e r f e r e in the
proposed procedure.
The m e t h o d is s i mple,
rapid,
precise,
a nd a c c u r a t e .
can be a p p l i e d to the d e t e r m i n a t i o n of m i c r o - q u a n t i t i e s of
n i t r i t e ion in a q u e o u s s o l u t i o n .
It
ABSORBANCE
56
FIG.1
ABSORBANCE AT 545 nm
57
58
TABLE - I
E^ffacts
aromatic primary amines and coupling reagents on
diazotisinq on colour intensity at 545nmx:
Primary
flrnmaf i n
aminesxx
%-# H i
Coupling Agemts xxx
—JLi
(i)
(ii)
I
0.020
0.008
II
0.011
III
(iii )
(iv)
(v)
0.007
0.035
0.080
0.008
0.009
0.036
0.110
0.210
0.044
0.008
0.034
0.365
IV
0.080
0.015
0.007
0.090
0.270
V
0.212
0.050
0.008
0.090
0.301
VI
0.250
0.070
0.006
0.052
0.396
VII
0.192
0.043
0.005
0.041
0.349
VIII
0.027
0.012
0.014
0.059
0.054
IX
0.201
0.069
0.006
0.069
0.365
X
0.184
0.020
0.011
0.074
0.306
XI
0.226
0.060
0.005
0.066
0.372
XII
0.182
0.048
0.008
0.041
0.372
: Each value is the average of five determinations
xx
: Primary aromatic amines : I=Anthralinic acid ;
II = p-Aminosalicylic acid; III = Benzocanine hydrochloride;
IV = p-Chloroaniline, V = o-Nitroaniline, VI = Procaine HC1;
VII = Sulfanilic acid; VIII = m-Aminophenol;
IX = Sulfamethoxazole; X = Metoclopromide HC1;
XI = Sulfanilamide; XII = p-Aminobenzoic acid
xxx : Coupling agents:
(i) 1-Naphthylamine; (ii) 2-Naphthylamine;
(iii) 1-Naphthol; (iv) 2-Naphthol (v) N-1-(Naphthyl)
ethylene diamine dihydrochloride.
59
0,60
0.55
0.50
-
ABSORBANCE AT 545 nm
0.45
FIG.3
EFFECT OF CONCENTRATION OF PROCAINE HCI.
60
TABLE - II
Effect of acids on the color intensity at 545nm* (O.D. )
Hydrochloric Acid
Sulfuric Acid
Acetic Acid
0.405
0.398
0.340
* Each value is average of five determinations.
K
O
ABSORBANCE AT 545 nm
61
0.1S
0,10
0 05
-L
0
O-T
10
1.5
JL
J.
2.0
2.5
3,0
3,5
4.0
HYDROCHLORIC ACID (ml;4N)
hlC.,4
EFFECT OF CONCENTRATION OF HYDROCHLORIC ACID
ABSORBANCE AT545 nm
62
63
64
o .io
oos
*
i
*
20
40
60
1
.. -
- ................. ...
80
100
1 120
140
TIME IN MIN,
FIG.7
EFFECT OF TIME OF REACTION
160
1M
ABSORBANCE AT 545 nm
65
FIG.S