Chapter 5

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Introduction

Understanding the effects of trace metals on human beings is very tedious, complex and it is
fascinating. The high concentrations may prove toxic and depletion may cause various metabolic
instabilities. In recent years awareness about the trace elements role, either beneficial or harmful
in human health has been increased. The alteration of concentration of trace elements in some
body fluids especially blood serum and plasma brings so many changes in human health[1].
Interest in trace elemental research in biology, environmental studies, toxicology, clinical
medicine and nutrition has become an exciting frontier. Among the trace elements zinc is an
essential element for human beings and different microorganisms. Most of the zinc (75- 80%) is
in erythrocytes the remaining is in plasma and leucosites[2-3 ].Zinc is associated as activator in
vital activities of flora and fauna by distributing in body fluids such as blood serum, milk and
also a micro nutrient in several enzymes. Several methods are found in the literature for the
determination of zinc in body fluids[4-12 ]. In the present investigation zinc was determined in
blood serum, milk Di amino di hydroxy pyrimidine as a selective complexing agent in the
conditions already established in our earlier communication [13-15].

Determination of Zn (II) in Milk,

Zinc is one of the important trace metal elements in the human body [16] .Zinc in milk powder is
also necessary for the healthy growth of children. Its content is directly related to human health
and infant growth and development. At present, methods for determination of zinc in milk
powder include atomic absorption spectroscopy [17,18] electrochemical analysis[19] , ICP-
AES5 , ICP-MS[20], catalytic kinetic spectrophotometry[21] , spectrophotometry [22-23] , etc.
In present study, it is noted that in presence of the surfactant gum Arabic, the Zn (II)-SCN– -
ERB (ERB = ethyl rhodamine-B) ternary system can form a rose red complex with a maximum
absorption at 625 nm in the acetic acid-sodium acetate medium of pH 5. The absorbance value
of the system has a linear relationship with the zinc concentration in the solution within a
particular range. A new ethyl rhodamine B spectrophotometry was thus established for rapid
determination of the trace zinc in milk powder.
Spectrophotometric Determination

Instrumentation

Elicomicro processor based double beam UV- visible spectrophotometer SL 210 equipped with 1
cm quartz cells were used for spectrophotometric measurements. The pH figure outed by using
Elico digital pH meter L.1 127 model.

Reagents and solutions

Required reagents obtained from Fisher Scientifics India.

Stock standard Zn(II) solution was prepared by dissolving 0.4397 g of Zn(II) sulphate hepta
hydrate in double distilled water containing 1000 μg/mL. The concentration of solution was
optimized by complex metric titration using EDTA. The working standard solutions were
prepared by suitable dilution of the stock solution.

Buffer solutions were prepared28 by employing 0.1 M Acetic acid, 0.1 M sodium acetate in the
pH range 2-8.

Solutions of diverse ions containing 1000 μg/mL were prepared by dissolving required amounts
of salts of the corresponding ions in double distilled water.

The reagent stock solution (0.1 M) was prepared by dissolving 1.421 g of DADHP in ethylene
glycol. This was adjusted to the suitable concentration by adding organic solvent.

Preparation of bidentate ligand

A bidentate ligand 2,5-Diamino-4,6- dihydroxypyrimidinewas prepared in three steps by the

methods reported in the literature25-26. Diethylmalonote (47.4 mL) was mixed with the mixture
of glacial acetic acid and water (1:1.5) and transferred in to 500 mL round bottom flask fitted
with mechanical stirrer and thermometer. The flask was cooled in an ice bath 0.65 g of NaNO2
0C
was added in portions by maintaining the temperature about 5 with continuous stirring. After
the addition of total NaNO2 the ice bath was removed stirring was continued for four hours. The
reaction mixture was extracted with ether and evaporated to procure diethyl- isonitrosomalonote.
Diethyl isonitrosomalonote mixed with1:3 mixture of acetic anhydride and glacial acetic acid in
a three necked round bottom flask fitted with mechanical stirrer, thermometer and dropping
funnel. 78.5 g of Zn dust was added in small portions by maintaining the temperature at 40-50
0C followed by intermittent cooling. The reaction mixture was filtered with suction, the filtrate
and washings were evaporated on the steam bath and then cooled in an ice bath by rapid stirring,
diethyl acetamido malonote separates out as a white crystalline solid. The yield was 35- 40 g,
M.P was (95-97 0C)(scheme-1).

Diethyl acetamido malonote was mixed with guanidinium carbonate and EtOH refluxed for 36 h,
then the reaction mixture was filtered by suction. The filtrate and the washings were evaporated
and cooled in an ice bath to separate out the 5- acetamido-2-amino-4,6- hydroxypyrimidine. This
was refluxed with 100 mL of 1 M HCl for 1 h then cooled in an ice bath filtered followed by
washings with HCl and acetone then air dried at 40 0C to procure the2,5-diamino-4,6-
dihydroxypyrimidine (25 g). The structure was confirmed from Mass, IR, H1 NMR Spectral
studies.

Spectro photometric measurements

Direct spectrophotometry

To achieve suitable environments for computation of Zn(II) required amounts collected with
sample covets and added 2 mL of pyridine and 1 mL of 2M HCl, then the pH was adjusted to 6
using acetic acid and sodium acetate buffer. The solutions are equilibrated with 5 mL of DADHP
(3 x 10-3 M) reagent solution and made up to 20 mL with double distilled water. The absorbance
of sample computed versus DADHP.

The composition of the complex was ensured by mole ratio, slope ratio and Job’s continuous
variation methods. The calibration plot was attained in in between 1-6 micrograms. Obeying the
Beer’s law.

Derivative spectrophotometry

To the aimed samples derivative spectra noted through group magnitude 9 as well as degree of
freedom 5 in the range 400-600 nm. The derivative peaks were measured by the peak zero
method at respective wave length. The peak heights were plotted against the amount of zinc to
obtain the calibration. The amount of zinc present in the pharmaceutical sample solutions were
computed from the calibration plots, both in direct and derivative spectrophotometry. The
calibration plots follow the straight line equationY=A+Bx where x is the concentration of
solution, Y is the measured absorbance or peak height, A is the intercept and B is the slope. By
substituting the experimental data in least square analysis, the equations were calculated as Y=-
0.01143+0.0309x for zero order spectrophotometry ∂A/∂λ=0.00396+0.00469x for first order
derivative, ∂2A/∂λ2 = 0.00329+ 0.00247x for second order derivative and ∂3A/∂λ3 =
0.00362+0.00200x for the third order derivative spectrophotometry respctively.
Result and discussion

Absorption spectra of DADHP and Zn(II)-DADHP complex

At the start absorption spectrum of the DADHP is recorded against the solvent blank. The
absorption spectrum ofZn(II) -DADHP complex is recorded against the ligand blank. The
absorption spectrum of both complex and ligandare shown in the fig.1. From the absorption
spectra, it is clear that the ligand shows maximum absorbance at 400-600nm, whereas the
complex shows maximum absorption at 480 nm.in acetic acid and sodium acetate buffer (Figure
1). Therefore, all the spectral measurements arecarried out at 480 nm.

Effect of PH:

Sizable delusions in the determination of zinc by atomic absorption spectrophotometry can be

caused by differences in pH among samples and standards. Zinc absorption is constant in the pH
range of about 2 to 5(fig:2). Between pH 5 and 10, a sharp decrease in absorption is observed.
With an increase above pH 10, zinc absorption levels off to a constant value. Experimental data
suggested that pH variation rather than chemical interferences is primarily responsible for the
variation of absorption in zinc analysis. Several possibilities, such as sorption of zinc to container
walls, zinc hydroxide formation, zinc complex formation, and zinc oxide formation in flame
subsequently attacked by hydrogen atoms, have been considered in an attempt to understand the
cause of pH-dependent zinc absorption.

Effect of salting out agent:

With reference to 5 mL aqueous solution contains 50 µg Zn2+ with different concentration of

KNO3 as salting out shaking with 5 mL of MS solution at 1×10−4 M concentration dissolved in
chloroform at time 15 minutes at the end separated the two layers and measure the absorbance of
Research Article organic layer at λmax=263 nm for Zn+2 against MS solution as blank.

Effect of reagent concentration/Effect metal ion concentration

A proximate 5 cc aqueous solution contains different concentration of Zn2+ with 0.3 M KNO3
for Zn2+ extracted according to general method. The results were as in (Fig3). The results show
optimum concentration of metal ion giving higher absorbance was 100 µg Zn2+ at this
concentration obtained the best rate for the forward direction .

Effect of diverse ions or Interferences effect

A precise 5 cc aqueous solution contains optimum quantity of Zn2+ at optimum conditions in

presence 0.1 M interferences, afterward replenishment extraction according to general method,
the results were as in Table 3. The results appear that there are clearly interferences for Co2+,
Ni2+, Fe3+, Hg2+ which are effected to decline extraction efficiency for Zn2+ and minimization
absorbance .

Shaking time effect

Extracted metal elements under study according to the procedure detailed in general method at
the optimum condition with different shaking time. The results were as in Figs. 4. The results
show optimum shaking time was 15 minutes for extraction Zn2+ in view of the fact that
solvation is un application of solvent extraction, and indirect method depend on thermodynamic
and kinetic laws whereas shaking time mean kinetic energy and 15 minutes as the optimum value
of shaking time .

Temperature effect

Extracted the metal cations under study according to general method at different temperature
and calculated D-value of extraction at each temperature. After that calculated extraction
constant Kex according to the relation below

D
K= n+ ¿
2+ ¿¿

M n +¿= Zn ¿
[ MS ] M ¿

Enhancing to 50°C as well as the lower value of ΔHex reflect the high nearly ions of ion pair
complex each one to other to form more stable contact ion pair or loose ion pair so that high
positive value of ΔSex reflect the mechanism of thermodynamic extraction depend on intropy
that is mean this method is intropic in region.
Effect of Light (Photo degradation).
The upshot of light is often considered an important factor in drug stability.The photodegradation
study was intended to obtain useful information about the sensitivity of the sample to light.
Conditions of irradiation were controlled to study the effect of light and not energy (heat). The
decomposition of irradiated solution with sunlight for about 6 hours was monitored using first
derivative spectrophotometry.The solution remained stable and there was no change neither in
spectrum maximum at 298nm nor in the concentration. This reflected the stability of solution
under these conditions.
Organic solvent effect

According to the general method at optimum condition extracted metal cations understudies by
use of different organic solvent differ in dielectric constant as a solvent for dissolved organic
reagent. The results were as in Table 4. The results in Table 4 demonstrated that there is not any
linear relation between dielectric constant of organic solvents used and D values that is mean
there is not any effect for polarity of organic solvents on the extraction according to solvation
method, but the results appear there is an effect for the structure of the organic solvent on
extraction efficiency giving higher distribution ratio (D) with amyl alcohol which is have
dielectric constant equal to (15.8) and the second organic solvent was chloroform which is have
dielectric constant equal to (4.806) at extraction Zn 2+. But with extraction Cd2+ illustrated
chloroform giving higher distribution ratio then 1,2-dichloroethane and benzene.

Composition of the complex

Bracing of experimental observations in mole ratio, Job’s continuous variation method and slope
ratio method confirms Zn(II) forms the 1:6 complex with the reagent and the stoichiometric ratio
is 1:3. So, it was confirmed that the reagent is a bidentate ligand, Figure 5.

Collection of Milk samples

Ten mothers who intended to feed for at least two years of post-partum with breast milk the
interest and goals of the study are informed in detailed and their consult was obtained. The milk
samples were also collected from the buffalo’s, cows and from dairy forms in and around the
Nellore town. 100 ml of milk was added drop wise to a heated crucible to evaporate with
frothing then heated strongly for one hour to remove the moisture. The dark ash obtained was
dissolved in the minimum of 1:1 Nitric acid and evaporated. The process was repeated for thrice
finally by adding dilute Hydrochloric acid to dried mass and filtered.

The filtrate was diluted to 100 ml, 2 ml of aliquots of the solution is used for the determination at
the pH 6.0. (Table.3.)In the human breast milk the zinc was quantified as 2.4-2.5 μg/ml which is
in good agreement with the reported values. Similarly in buffalo’s, cow’s and pasteurized milk
was found 3.5-5.1 μg/ml. Milk represents the most suitable pattern of nutrients to need the
physiological requirements in young infants. Hence an accurate and complete knowledge of the
composition of the human milk and other sources of milk is essential and adequate for the
growth of infants and also to develop adequately defined formulas to be used as a substitute for
human milk. So our work is alarming the usage of breast milk and synthesis of milk products for
the growth of neonates and infants.

Analisys

To scrutinize the applicability of Beer’s law for the present system, the following procedure is
adopted. An aliquots of standard solutions containing microgram quantities of Zn(II) are taken in
a series of ten 20 ml comparison tubes followed by the addition of 2 ml of pyridine and 1 ml of
2M HCl, the pH was adjusted to 6.0 using acetic acid and sodium acetate buffer. To this 5 ml of
Di Amino Dihydroxy Pyrimidine (3x10- 3M) solution are added and the contents are finally
made upto the mark with double distilled water. The absorbance of the solutions is measured at
480nm against the reagent blank prepared under identical conditions .It was found that Beer’s
law is obeyed in the range 0.5-6.0 μg /ml. Further for the above solutions the derivative spectras
were also recorded with group size 9 and degree of freedom 5 in the wave length range 400-
600nm. The derivative peaks were measured by the peak zero method at respective wave length.
The peak heights were plotted against the amount of zinc gives a linear plot indicating the
applicability of Beer’s law in the range 0.5-6.0 μg /ml. The performance of the calibration plot
has been verified for ten replicate determinations.(Table.1.Fig.1)
Determination Blood Serum

Zinc (II) has been determined spectrophotometrically by using different heterocyclic azo dyes. A
new heterocyclic azo dye, Bis-[2,6-(2´-hydroxy-4´-sulpho-1´-napthylazo)] pyridine disodium
salt, HSNP has been synthesized by barman et al. [24] and has been successfully used for
spectrophotometric determination of nickel5 and zinc in pharmaceutical samples [25]. The
review of literatures indicates that not much attention has been paid for the spectrophotometric
determination of zinc (II) in biological samples . This has promoted the researchers to make a
systematic investigation for utilizing bis-[2,6-(2′-hydroxy-4′-sulpho-1′- napthylazo)]pyridine
disodium salt for the first time for spectrophotometric determination of zinc (II) in ppm level.
The established method is successfully applied for the spectrophotometric determination of zinc
(II) in human blood serum of diabetic patients. The proposed method when compared with other
spectrophotometric methods was found to be quite sensitive and selective. It also offers
advantages like realiability and reproducibility in addition to its simplicity, instant colour
development and less interference.

Itnstrumentation

Elicomicro processor based double beam UV- visible spectrophotometer SL 210 equipped with 1
cm quartz cells were used for spectrophotometric measurements. The pH calibrations
complained by using Elico digital pH meter L.1 127 model.

Reagents and solutions

Essential reagents obtained from Fisher Scientifics India.

A primary standard Zn(II) solution was prepared by dissolving 0.4397 g of Zn(II) sulphate hepta
hydrate in double distilled water containing 1000 μg/mL. The concentration of solution was
optimized by complexometric titration using EDTA. The working standard solutions were
prepared by suitable dilution of the stock solution.

Buffer solutions were prepared28 by employing 0.1 M Acetic acid, 0.1 M sodium acetate in the
pH range 2-8.
Solutions of diverse ions containing 1000 μg/mL were prepared by dissolving required amounts
of salts of the corresponding ions in double distilled water.

The reagent stock solution (0.1 M) was prepared by dissolving 1.421 g of DADHP in ethylene
glycol. This was adjusted to the suitable concentrationby adding organic solvent.

Preparation of DADHP ligand

2,5-Diamino-4,6- dihydroxypyrimidinewas prepared in three steps by the methods reported in

the literature25-26. Diethylmalonote (47.4 mL) was mixed with the mixture of glacial acetic acid
and water (1:1.5) and transferred in to 500 mL round bottom flask fitted with mechanical stirrer
and thermometer. The flask was cooled in an ice bath 0.65 g of NaNO2 was added in portions by
maintaining the temperature about 5 0C with continuous stirring. After the addition of total
NaNO2 the ice bath was removed stirring was continued for four hours. The reaction mixture
was extracted with ether and evaporated to procure diethyl- isonitrosomalonote.

Diethyl isonitrosomalonote mixed with1:3 mixture of acetic anhydride and glacial acetic acid in
a three necked round bottom flask fitted with mechanical stirrer, thermometer and dropping
funnel. 78.5 g of Zn dust was added in small portions by maintaining the temperature at 40-50
0C followed by intermittent cooling. The reaction mixture was filtered with suction, the filtrate
and washings were evaporated on the steam bath and then cooled in an ice bath by rapid stirring,
diethyl acetamido malonote separates out as a white crystalline solid. The yield was 35- 40 g,
M.P was (95-97 0C)(scheme-1).

Diethyl acetamido malonote was mixed with guanidinium carbonate and EtOH refluxed for 36 h,
then the reaction mixture was filtered by suction. The filtrate and the washings were evaporated
and cooled in an ice bath to separate out the 5- acetamido-2-amino-4,6- hydroxypyrimidine. This
was refluxed with 100 mL of 1 M HCl for 1 h then cooled in an ice bath filtered followed by
washings with HCl and acetone then air dried at 40 0C to procure the2,5-diamino-4,6-
dihydroxypyrimidine (25 g). The structure was confirmed from Mass, IR, H1 NMR Spectral
studies.
Spectrophotometric measurements

Direct spectrophotometry

To obtain suitable environmentsto computation of Zn(II) required amounts collectedwit sample

covets and added 2 mL of pyridine and 1 mL of 2M HCl, then the pH was adjusted to 6 using
acetic acid and sodium acetate buffer. The solutions are equilibrated with 5 mL of DADHP (3 x
10-3 M) reagent solution and made up to 20 mL with double distilled water. The absorbance of
sample computed versusDADHP.

The composition of the complex was ensured by mole ratio, slope ratio and Job’s continuous
variation methods. The calibration plot was attained in in between 1-6 micrograms. obeying the
Beer’s law.

Derivative spectrophotometry

To the aimed samples derivative spectra notedthrough group magnitude 9 as well as degree of
freedom 5 in the range 400-600 nm. The derivative peaks were measured by the peak zero
method at respective wave length. The peak heights were plotted against the amount of zinc to
obtain the calibration. The amount of zinc present in the pharmaceutical sample solutions were
computed from the calibration plots, both in direct and derivative spectrophotometry. The
calibration plots follow the straight line equationY=A+Bx where x is the concentration of
solution, Y is the measured absorbance or peak height, A is the intercept and B is the slope. By
substituting the experimental data in least square analysis, the equations were calculated as Y=-
0.01143+0.0309x for zero order spectrophotometry ∂A/∂λ=0.00396+0.00469x for first order
derivative, ∂2A/∂λ2 = 0.00329+ 0.00247x for second order derivative and ∂3A/∂λ3 =
0.00362+0.00200x for the third order derivative spectrophotometry respctively.

Result and discussion

Absorption spectra of DADHP and Zn(II)-DADHP complex

Initially absorption spectrum of the DADHP is recorded against the solvent blank. The
absorption spectrum of Zn(II) -DADHP complex is recorded against the ligand blank. The
absorption spectrum of both complex and ligandare shown in the fig.1. From the absorption
spectra, it is clear that the ligand shows maximum absorbance at 400-600nm, whereas the
complex shows maximum absorption at 480 nm.in acetic acid and sodium acetate buffer (Figure
1). Therefore, all the spectral measurements are carried out at 480 nm.

Effect of PH:

Substantial errors in the determination of zinc by atomic absorption spectrophotometry can be

caused by differences in pH among samples and standards. Zinc absorption is constant in the pH
range of about 2 to 5(fig:2). Between pH 5 and 10, a sharp decrease in absorption is observed.
With an increase above pH 10, zinc absorption levels off to a constant value. Experimental data
suggested that pH variation rather than chemical interferences is primarily responsible for the
variation of absorption in zinc analysis. Several possibilities, such as sorption of zinc to container
walls, zinc hydroxide formation, zinc complex formation, and zinc oxide formation in flame
subsequently attacked by hydrogen atoms, have been considered in an attempt to understand the
cause of pH-dependent zinc absorption.
Effect of salting out agent:

About 5 mL aqueous solution contains 50 µg Zn2+ with different concentration of KNO3 as

salting out shaking with 5 mL of MS solution at 1×10−4 M concentration dissolved in
chloroform at time 15 minutes at the end separated the two layers and measure the absorbance of
Research Article organic layer at λmax=263 nm for Zn+2 against MS solution as blank.

Effect of reagent concentration/Effect metal ion concentration

About 5 mL aqueous solution contains different concentration of Zn2+ with 0.3 M KNO3 for
Zn2+ extracted according to general method. The results were as in (Fig3). The results show
optimum concentration of metal ion giving higher absorbance was 100 µg Zn2+ at this
concentration obtained the best rate for the forward direction.

Effect of diverse ions or Interferences effect

About 5 mL aqueous solution contains optimum quantity of Zn2+ at optimum conditions in

presence 0.1 M interferences, afterward replenishment extraction according to general method,
the results were as in Table 3. The results appear that there are clearly interferences for Co2+,
Ni2+, Fe3+, Hg2+ which are effected to decline extraction efficiency for Zn2+ and minimization
absorbance .

Shaking time effect

Extracted metal elements under study according to the procedure detailed in general method at
the optimum condition with different shaking time. The results were as in Figs. 4. The results
show optimum shaking time was 15 minutes for extraction Zn2+ in view of the fact that
solvation is un application of solvent extraction, and indirect method depend on thermodynamic
and kinetic laws whereas shaking time mean kinetic energy and 15 minutes as the optimum value
of shaking time .

Temperature effect

Extracted the metal cations under study according to general method at different temperature
and calculated D-value of extraction at each temperature. After that calculated extraction
constant Kex according to the relation below
 D
K= n+ ¿
2+ ¿¿

M n +¿= Zn ¿
[ MS ] M ¿

increasing to 50°C as well as the lower value of ΔHex reflect the high nearly ions of ion pair
complex each one to other to form more stable contact ion pair or loose ion pair so that high
positive value of ΔSex reflect the mechanism of thermodynamic extraction depend on intropy
that is mean this method is intropic in region.

Effect of Light (Photo degradation).

The effect of lightis often considered an important factor in drug stability.The photodegradation
study was intended to obtain useful information about the sensitivity of the sample to light.
Conditions of irradiation were controlled to study the effect of light and not energy (heat). The
decomposition of irradiated solution with sunlight for about 6 hours was monitored using first
derivative spectrophotometry.The solution remained stable and there was no change neither in
spectrum maximum at 298nm nor in the concentration. This reflected the stability of solution
under these conditions.
Organic solvent effect

According to the general method at optimum condition extracted metal cations understudies by
use of different organic solvent differ in dielectric constant as a solvent for dissolved organic
reagent. The results were as in Table 4. The results in Table 4 demonstrated that there is not any
linear relation between dielectric constant of organic solvents used and D values that is mean
there is not any effect for polarity of organic solvents on the extraction according to solvation
method, but the results appear there is an effect for the structure of the organic solvent on
extraction efficiency giving higher distribution ratio (D) with amyl alcohol which is have
dielectric constant equal to (15.8) and the second organic solvent was chloroform which is have
dielectric constant equal to (4.806) at extraction Zn2+.

Composition of the complex

Enlivening of experimental interpretation in mole ratio, Job’s continuous variation method and
slope ratio method confirms Zn(II) forms the 1:6 complex with the reagent and the
stoichiometric ratio is 1:3. So, it was confirmed that the reagent is a bidentate ligand, Figure 5.
Collection of blood samples

A total of 5 ml of blood was collected in a sterilic plastic test tube with screw cap from people
of different age groups of normal and diabetic patients in Nellore town with the help of Jaya
Bharat hospital clinical laboratories. Approximately 5 ml of drawn blood taken into centrifuged
tubes and allowed to stand for 30 minutes then centrifuged at 3000 RPM for about 10 minutes.
The serum separated is decanted. The total serum was treated with 1 ml of 20% Tri Chloro
Acetic Acid (TCA) for the deprotinisation, then the sample is allowed for quantification of
zinc.The zinc content in the blood sample of different aged people in normal health conditions
and suffering with diabetic was found to be 2.8-4 μg/ml and 1.15-1.5 μg/ml respectively. These
values clearly indicating the important role of zinc deficiency in diabetic patients.

The determination of the circulating levels of zinc in serum has been most widely used approach
for the assessment of zinc nutrition, metabolic states, stress, infection; food intake shorter fasting
and hormonal state all appear to influence. The decreased zinc concentration in serum decreases
its excretion from the breast milk by several mediators because mammary glands gets zinc from
the blood . Further the decrease zinc concentration causes the leukemia.

Analisys

To examine the applicability of Beer’s law for the present system, the following procedure is
adopted. An aliquots of standard solutions containing microgram quantities of Zn(II) are taken in
a series of ten 20 ml comparison tubes followed by the addition of 2 ml of pyridine and 1 ml of
2M HCl, the pH was adjusted to 6.0 using acetic acid and sodium acetate buffer. To this 5 ml of
Di Amino Dihydroxy Pyrimidine (3x10- 3M) solution are added and the contents are finally
made upto the mark with double distilled water. The absorbance of the solutions is measured at
480nm against the reagent blank prepared under identical conditions .It was found that Beer’s
law is obeyed in the range 0.5-6.0 μg /ml. Further for the above solutions the derivative spectras
were also recorded with group size 9 and degree of freedom 5 in the wave length range 400-
600nm. The derivative peaks were measured by the peak zero method at respective wave length.
The peak heights were plotted against the amount of zinc gives a linear plot indicating the
applicability of Beer’s law in the range 0.5-6.0 μg /ml. The performance of the calibration plot
has been verified for ten replicate determinations.(Table.1.Fig.1).
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