123

CHAPTER 5

SPECTROPHOTOMETRIC DETERMINATION OF CHROMIUM USING

TOLUIDINE BLUE AND SAFRANINE O AS NEW REAGENTS

5.1 INTRODUCTION

5.2 ANALYTICAL CHEMISTRY

5.3 APPARATUS

5.4 REAGENTS AND SOLUTIONS

5.5 PROCEDURES

5.6 RESULTS AND DISCUSSION

5.7 APPLICATIONS

5.8 CONCLUSIONS

5.9 REFERENCES

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5.1 INTRODUCTION

Nicolas-Louis Vauquelin discovered chromium in 1797 in Siberian red lead,

the mineral crocoite, PbCrO4. In 1798 he isolated the new metal by reduction of CrO3

with charcoal at high temperature [1,2]. In the same year he analyzed a Peruvian

emrald and found that its green color is due to the new element. Fourcroy and Huay

suggested the name chromium (from the Greek chroma, color) for the new element

because of its many colored compounds [3]. In 1798 Tobias Lowitz and Martin

Heinrich Klaproth independently found chromium in chromite samples from Russia

and in the following year Tassaert, a German Chemist at the Paris school of Mines

found it in French chromite. This ore, a spinel, Fe(CrO2)2 is the only commercial

sources of chromium. Chromium metal was obtained by Moissan in 1893 by

reduction of chromic oxide with carbon in an electric furnance. In 1894 Goldschmidt

developed the alumino-thermit process for producing chromium by the reduction of

oxide with aluminium powder [4]. The higher grades of ore contain 42-56 % Cr2O3,

10-26 % Fe and varying amounts of other substances such as magnesia, alumina and

silica.

Chromium is the recently recognized biologically essential trace metal. The

first conclusive evidence demonstrating a metabolic role of chromium was obtained

by Mertz and Schwarz in a series of investigations of which the first report appeared

in 1955 [5]. Chromium is found in the body in very low concentrations, with the total

chromium level in the adult human body being about six grams. The highest levels of

chromium are found in the liver, kidney, spleen and bone. The chromium that our

body requires is called trivalent chromium. Chromium helps to improve the body's

responses to the hormone insulin. Insulin is an important hormone for controlling

blood sugar levels as well as for metabolizing fats and proteins in the body.

Chromium deficiency can cause the body to overproduce insulin but the body is

unable to respond to this insulin. This is called insulin resistance and it is a feature of

type-II diabetes. It was once thought that chromium could assist in fat loss and help to

maintain lean body mass (muscle), but these effects are not seen in humans, only in

experimental animals. The recommended daily intake of chromium is 50 to 100

micrograms (0.05-0.1 mg). Various forms of chromium are available at pharmacies

and health food shops. Chromium picolinate may be the one that is best absorbed into

125

the body [6]. Very little chromium in the form of inorganic compounds, such as

chromic chloride is absorbed. The efficiency of absorption of chromium from chromic

chloride is less than 2 % [7]. The efficiency of absorption of chromium from organic

compounds is higher. For example, approximately 2.8 % of an injected dose of

chromium picolinate is absorbed. Following absorption, chromium is bound to

transferrin and albumin and chromium is transported primarily by transferrin. The

evidence which implicates chromium as a critical cofactor in the action of insulin. It

has been variously suggested that chromium enhances insulin-binding, insulin

receptor number, insulin internalization and β-cell sensitivity [8]. Such findings

establish a link between chromium and diabetes. The evidence comes from the studies

conducted with a patient receiving Total Parenteral Nutrition (TPN), who developed

severe signs of diabetes, including weight loss and hyperglycemia that was refactory

to increasing insulin dosage [9].

The two most impotant functions of chromium in steels are improving the

mechanical properties particularly hardenability and increasing the corrosion

resistance [10]. The magnitude of the effect in each case is roughly proportional to the

percentage of chromium in the steel. Low chromium steels (< 3 % of Cr) produced in

all structural shapes such as bars, tubes, sheets, plates, etc., are used extensively as

engineering materials in every branch of industry. For all but the heaviest duty

applications, chromium content is generally less than 6 %. Steels contain more than

10 % chromium and are designated stainless because of their resistance to corrosion

and oxidation. Non-hardenable grade contain 0.08-0.20 % carbon and 11.25-27.0 %

chromium. Type 430 (AISI) is used in large quantities for trim on buildings,

automobiles, etc., and for nitric acid manufacturing equipment. The austenitic

stainless steels (non-hardenable) contain 16-26 % of chromium and 0.15-1.25 %

carbon.

Chromium deficiency can cause insulin resistance and hyperglycaemia (high

blood glucose levels that cannot be controlled by insulin), cardiovascular disease and

elevated fat levels in the blood. Severe chromium deficiency may cause weight loss,

poor coordination, destruction of the nerves in the extremities of the body (peripheral

neuropathy) and inflammation of the brain. The effects of excessive dietary chromium

126

are not well known. Some forms of chromium that are found in the environment may

be cancer causing, but this type of chromium is different from dietary chromium.

Food with high chromium content are fruits, vegetables, whole grains (e.g. oats and

barley), seeds, nuts, legumes (peas and beans) and brewer's yeast. When these food is

processed, particularly using stainless steel equipment (e.g. when cooking or

canning), their chromium content may increase. Allergy to chromium compounds

carries in men, a worse prognosis than dose sensitization to other allergens. The

reason is not known. Continued contact with unrecognized chromium compounds in

the environment or possibly ingestion of chromium compounds has been considered

as possible explanations [11].

The determination of micro amounts of chromium in soils and other naturally

occurring materials are of considerable interest because of the contrasting biological

effects of its two common oxidation states, chromium(III) and chromium(VI) and also

the growing interest in environmental problems. It is known that an increase in the

content in soils makes them infertile and toxic effect depends to some extent on the

chromium oxidation state. On the other hand, the introduction of chromium salts into

soils has some positive effects due to activation of some biochemical processes [12].

Chromium(III) is an essential nutrient for maintaining normal physiological function,

where as chromium(VI) is toxic [13].

5.2 ANALYTICAL CHEMISTRY

Many methods have been reported for the quantitative determination of

chromium. The analytical technique varies from inductively coupled plasma-atomic

emission spectroscopy [14], atomic absorption spectroscopy [15], neutron activation

analysis [16], X-ray absorption spectroscopy [17], complexometric [18], catalytic-

kinetic [19], sequential injection [20] to flow injection methods [21,22].

A survey of literature revealed that a large number of reagents are suitable for

the spectrophotometric determination of chromium. One of the spectrophotometric

method was based on the color of the [Cr(C2O4)]3- ion [23]. Cahnmann and Ruth

reported a spectrophotometric method for the determination of chromium using

127

1,5-diphenylcarbazide reagent [24]. In this method Beckman DU spectrophotometer

was used with a wave length of 543 nm and the reaction was sensitive to 0.005 ppm.

Selmer-Olsen used Complexon-IV as a reagent for the determination of

chromium [25]. Chromium(III) formed a stable water soluble violet complex with

Complexon-IV. The 1:1 chromium complex exhibited absorption maxima at 395 and

540 nm. Beer’s law was obeyed in the range 2-�������-1 of chromium.

Dwight and John used diphenylcarbazide as a spectrophotometric reagent for

the determination of chromium in human plasma and red blood cells [26]. Traces of

chromium in human blood was determined by a method which utilized the red-violet

complex formed by the reaction of Cr2O72- with diphenylcarbazide. The concentration

of chromium in normal human plasma ranged from 0.017 to 0.052 ppm, which was in

good agreement with previously reported values. The concentration of chromium in

red cells ranged from 0.014-0.038 ppm. Standard deviation by the method was 1.6 %.

8-Hydroxyquinaldine [27] was also used as a spectrophotometric reagent for the

determination of chromium in uranium.

Sumio and Koji used 1-phenylthiosemicarbazide as a reagent for the

spectrophotometric determination of chromium [28]. Chromium formed a brown

color with 1-phenylthiosemicarbazide. Den Boef and Poeder summarized the

interferences in the spectrophotometric determination of chromium(III) with pyridine-

2,6-dicarbonic acid [29,30]. Results were obtained in the determination of Cr(III) in

presence of a 200-fold excess of Cu, Ni, Al, Mn, Zn or Fe(III) and a 100-fold excess

of cobalt. A selective spectrophotometric determinations of chromium(III) with

complexans was described [30]. The method was based on the fact that the

chromium(IIl) complexes were formed rapidly at boiling temperature, but very slow

at room temperature, while the formation of some interfering complexes took place

instantaneously. Determinations with EDTA were more sensitive, but the combined

presence of cobalt and other metals interfered. The combined presence of a l00-fold

amount of copper, nickel, cobalt and iron generally has no effect on the results.

Takao et al. described a extraction-spectrophotometric determination of

chromium(III) with 4-(2-pyridylazo)-resorcinol (PAR) [31]. PAR(H2R) formed a 1:3

128

complex with chromium(III) in a acetate buffer solution at pH 5. The complex formed

an ion-associated compound with tetradecyldimethylbenzylammonium ion which was

extracted into chloroform, the molar absorptivity was 4.7×104 at 540 nm. EDTA was

added as a masking agent after the Cr(HR)3 was formed. Iron, cobalt and nickel

interfered seriously and the method was made specific for chromium by a preliminary

extraction of these metals with cupferron. The sensitivity of the method was seven

times higher than that of the diphenylcarbazide method. Koichi et al. reported xylenol

orange as a reagent for the spectrophotometric determination of chromium [32].

Einar and Walter described a spectrophotormetric determination of chromium

with thioglycollic acid [33]. Cheng reported a simple and sensitive method for the

spectrophotometric determination of chromium with xylenol orange and

methylthymol blue [34]. Xylenol orange and chromium(III) formed a red colored

complex at pH 3 on heating for 20 minutes. The molar absorptivity was 19.0×103

Lmol�cm�. Methylthymol blue was a less sensitive reagent for chromium; the molar

absorptivity was 11.5×103 Lmol�cm�.

Johnston and Holland reported a visible spectrophotometric method for the

determination of chromium(III) with 3-thianaphthenoyltrifluoroacetone at a pH of 4.0

to 4.5 [35]. The effects due to pH, time, solvents, reagent concentration and diverse

ions were reported. Beer's law was obeyed and the molar extinction coefficient is

1.3×103 Lmol�cm�. Katsuya and Yukiteru reportd 2-hydroxy-1-(2-hydroxy-4-sulfo-

1-naphthylazo)naphthalein reagent for the spectrophotometric determination of

chromium [36].

Ferng and Parker described the ternary complex of chromium- peroxo-4-(2-

pyridylazo)resorcinol which exhibited an apparent molar absorptivity of 6.280×103

Lmol–1cm–1 when extracted into ethyl acetate from 0.1 M sulfuric acid solution for the

determination of chromium [37]. Beer's law was valid up to 6.0 µgmL–1 of chromium.

Conditions for optimum color formation, complex composition, effect of diverse ions

and application to the determination of chromium in steels were described. Li and

Hercules described a method for the determination of chromium in biological samples

by chemiluminescence [38].

129

Kumpulainen et al. reported a method for the determination of chromium in

selected United States Diets [39]. Rizvi used tropolone as a reagent for the

spectrophotometric determination of chromium [40]. Chromium formed a golden

yellow colored complex with tropolone on heating on a water bath; the colored

moiety was extracted to CHCl3. The complex exhibited maximum absorbance at 400

nm.

Reddy and Reddy used cyclohexane-1,3-dionebisthiosemicarbazone

monohydrochloride for the rapid spectrophotometric determination of chromium(VI)

[41]. Cyclohexane-1,3-dionebisthiosemicarbazone monohydrochloride produced

yellow colored solutions with Cr(VI) in NaOAc-HCl medium. Molar absorptivity

value of the system was 1.21×104 Lmol-1cm-1 at 370 nm.

Gowda and Raj reported fluphenazine hydrochloride as a reagent for the

determination of chromium [42]. Fluphenazine hydrochloride formed a red colored

species with chromium instantaneously at room temperature in 2.0-5.5 M H3PO4

medium. The red colored species exhibited maximum absorbance at 500 nm with

molar absorptivity of 2.616×104 Lmol-1cm-1. Beer's law was valid over the

concentration range 0.05-1.85 ppm of chromium.

Falian and Tao used dibromoalizarin violet for the determination of chromium

in waste water [43]. Chromium reacted with dibromoalizarin violet at pH 5 (using

hexamine-HCl buffer) in the presence of cetyltrimethylammonium bromide at

85-90°C in 30 minutes, formed a blue-green complex. The molar absorptivity of the

complex was 4.54×104 Lmol-1cm-1 at 620 nm. Beer's law was obeyed at 0-������-1

of chromium in 25 mL.

Bin reported a spectrophotometric determination of chromium using

salicylfluorone in phosphoric acid medium [44]. Chromium reacted with

salicylfluorone reagent to form a yellow colored complex with molar absorptivity of

2.86×104 Lmol-1cm-1 at 490 nm. Beer’s law was obeyed in the range 0-50 ��� ���

chromium in 25 mL.

130

Fanqian reported acid chrome blue-K as a new reagent for the

spectrophotometric determination of chromium [45]. The reagent formed a colored

complex with chromium(III). The molar absorptivity was 2.0×104 Lmol-1cm-1 and

Sandell’s sensitivity 2.2×10-3��� �-2 at 597 nm. Beer's law was obeyed in the range

0-������-1 of chromium in 50 mL. Strubel and Rzepka-Glinder described a method

for the analysis of chromium in saliva by atomic absorption spectrometry [46].

Lisheng and Zhaoping used aluminon for the spectrophotometric

determination of micro amounts of chromium in steels [47]. Chromium was

determined in steel by measuring the absorbance at 545 nm of the complex formed by

the reaction of Cr(VI) with aluminon at pH 3.3-4.2 in HOAc-NaOAc buffer solution.

The molar absorptivity of the complex was 1.9×104 Lmol-1cm-1. Beer's law was

obeyed in the range 5-�������-1 of chromium in 50 mL. The complex was stable for

about 4 hours. Sodium diethyldithiocarbamate [48], benzyl-tributylammonium [49]

were also reported as spectrophotometric reagents for the determination of chromium

in waste water and steels.

Ram et al. used malachite green [50] for the spectrophotometric determination

of chromium in waste water. The reagent formed a green colored complex with

chromium in acetic acid at pH 2.5. The molar absorptivity of the system was 8.0×104

Lmol-1cm-1 at 560 nm.

Kamburova described a spectrophotometric determination of chromium with

iodonitrotetrazolium chloride and tetrazolium violet [51-53]. The method was based

on the interaction of iodonitrotetrazolium chloride and tetrazolium violet with

chromium(VI) and the formation of ion-associates with a 1:1 composition in

hydrochloric acid medium. Spectrophotometric determination of chromium(VI) with

methylene blue was developed [52]. In this method the interaction of Cr(VI) and the

methylene blue was examined. The ion-associate formed was extracted into

1,2-dichlorethane. The optimum conditions was established and the values obtained

for the conditional extraction constant K�ex, distribution constant K�D and association

�������� ���� ���� ������� ���� �������� ���� ���� �������������� ��� �� ��� ��������� ���

Cr(VI) in soils and alloys. Kamburova described triphenyltetrazolium chloride as a

reagent for the spectrophotometric determination of chromium(VI) [53]. Bokic et al.

131

reported a spectrophotometric determination of chromium with diantipyryl(3,4-

dioxomethenyl) phenylmethane [54].

Arya and Bansal described a spectrophotometric determination of

chromium(VI) with ferron [55]. Chromium(VI) formed a pink coloured solution in

chloroform in the presence of ferron when extracted from slightly acidic medium. The

reaction was measured at 510 nm. Beers law was obeyed in the range of 5–70 µgmL-1.

Most of the important metal ions do not interfered. The relative standard deviation

was 2.78 %. Baluja-Santos et al. described spectrophotometric determination of

chromium(VI) with N-(2-Acetamido)iminodiacetic acid (ADA) [56].

Sasaki et al. reported extraction spectrophotometric determination of

chromium(VI) [57]. The method was based on the reaction of chromium(VI) with

o,o'-dibutyl dithiophosphate ion, which formed tris(dibutyl

dithiophosphato)chromium(III) and tetrabutyl thiophosphoryl disulfide in an acidic

solution (pH 1.2-1.7). Both the products were extracted into hexane and the

absorbance was measured at 278 nm. The chromium(III) complex corresponding to

two-thirds of the initial chromium(VI) concentration and the disulfide corresponding

to three-halves of the initial chromium(VI) concentration were extracted under the

optimum conditions.

El-Sayed and Abd-Elmottaleb described a fourth derivative

spectrophotometric determination of chromium(III) with eriochrome cyanine R (ECR)

[58]. The method was based on the fourth derivative value (D4) at 545 nm. The

experimental and instrumental variables (wavelength range, scan speed, band width

and order of derivative) were optimized. The molar absorptivity was 3.75×105 with a

relative standard deviation of 1.19 %. The method was valid for concentrations

between 20 ngmL-1 and 80 ngmL-1 of chromium(III). The molar ratio of the formed

complex was 1:2 (M:L). The proposed method was successfully applied to the

determination of chromium in steels.

Raj and Gowda reported thioridazine hydrochloride as a sensitive and

selective reagent for the spectrophotometric determination of chromium [59]. The

132

reagent formed a blue coloured radical cation with chromium(VI) instantaneously at

room temperature in 1–4 M orthophosphoric acid medium. The blue colored species

exhibited an absorption maximum at 640 nm with a molar absorption coefficient of

2.577×104 Lmol–1cm–1. A 27-fold molar excess of the reagent was necessary for the

development of the maximum colour intensity. Beer's law was obeyed over the

concentration range 0.05–2 ppm of chromium(VI) with an optimum concentration

range of 0.2–1.6 ppm. The effects of acidity, time, temperature, order of addition of

reagents, reagent concentration and the tolerance limit of the method towards various

cations and anions associated with chromium were reported. The method was used

successfully for the determination of chromium in chromium steels.

Rosa et al. reported a sensitive spectrophotometric determination of chromium

with 2-(5-chloro-2-pyridylazo)-5-dimethylaminophenol [60]. A mixture of

hydroethanolic solution of the reagent and an aqueous solution of Cr(III) were heated.

Three different complex species were formed depending on the composition and pH

of the medium (ε=1.7×105 Lmol-1cm-1). The stoichiometry of the different complexes

was determined, the formation mechanisms were elucidated and the respective

constants were calculated. A useful spectrophotometric method was proposed for

Cr(III) in concentrations ranged from 15 to 400 ppb. The proper ways to reduce

interferences produced by Fe(III), Nb(V), Ta(V), Ti(IV), V(V), Co(II), Zr(IV), Sn(II),

Al(III), Mn(II) and Cu(II) were described. The methods were applied to chromium

determination in water samples with satisfactory results.

Burns and Dunford described a spectrophotometric determination of

chromium(VI) by extraction of protriptylinium dichromate [61]. In this method

chromium(VI) was determined spectrophotometrically at 365 nm after its extraction

as protriptylinium dichromate into acetone-chloroform (1:1, v/v). The effects of

acidity, diverse ions and masking studies were reported. The relative standard

��!������� ���� �������������� ��� "��� ��� ��� �� �������� ���� ��#� $�� ���� �%����� ����

applied to the spectrophotometric determination of chromium in steels. Maheswari

and Balasubramanian reported rhodamine-6G as a spectrophotometric reagent for the

determination of chromium [62].

133

Wrobel et al. repored enhanced spectrophotometric determination of

chromium(VI) with diphenylcarbazide using internal standard and derivative

spectrophotometry [63]. The following procedure was used: (1) addition of internal

standard and formation of ion pairs of Cr(VI) with benzyltributylammonium bromide

(BTAB) (sample volume 100 mL), (2) extraction to 10 mL of methylene chloride, (3)

evaporation in nitrogen stream, and (4) redissolution in a micro-volume with addition

of diphenylcarbazide for color developm���� &������ !�� ��� "��� ��'�� ����

preconcentration factor achieved was about 400 and it was shown that using internal

standard, the analytical errors due to sample treatment were reduced. The analytical

signals for chromium and internal standard were obtained at 591.30 and 653.50 nm.

���� ����%�� ��� ���� ������� �� �!�� ����� ����(� ���� ����� ������ )� ���*� ���,

� ������ ������ ������ )� ���#� ���+� ��� ������ ���� �� ���� �,�"�$+� ���� ���� ��� ���

3.2%, correlation coefficient of linear regression was 0.9985. The proposed procedure

was applied to determination of chromium(VI) in tap water. Total chromium was

determined by electrothermal atomic absorption spectrometry, the recovery of

hexavalent chromium added was then evaluated and compared with the results of the

proposed procedure. In this experiment, good agreement was obtained between results

obtained by the two methods.

Hoshi et al. described a simple and rapid spectrophotometric determination of

trace amounts of chromium(VI) after preconcentration as its colored complex on

chitin [64]. The chromium(VI) was collected as its 1,5-diphenylcarbazide(DPC)

complex on a column of chitin in the presence of dodecyl sulfate as counter-ion. The

Cr-DPC complex retained on the chitin was eluted with a mixture of methanol:acetic

acid(1M) (7:3, v/v) and the absorbance of the eluent was measured at 541 nm. Beer’s

law was obeyed over the concentration range of 0.05–��*� ��� ��� ����� �&-.'� ����������

1 mL of the eluent. The apparent molar absorptivity was 3.5×104 Lmol�cm�. The

tolerance limits for Fe(III) was low, i.e. ten times that of chromium(VI), but some

metal ions and common inorganic anions did not interfere in the concentration range

of 100–10000 times that of chromium(VI). The method was applied to the

determination of chromium(VI) in natural water samples.

Ressalan et al. reported 3-hydroxy-3-phenyl-1-o-hydroxyphenyltriazene,

3-hydroxy-3-p-tolyl-1-o-nitrophenyl-triazene and 3-hydroxy-3-phenyl-1-o-

134

carboxyphenyl-triazene as reagents for the spectrophotometric determination of

chromium [65-67].

Balogh et al. reported a spectrophotometric study of the complexation and

extraction of chromium(VI) with Cyanine dyes [68]. The molar absorptivities of the

ion associates were in the range of 2.5–3.6×105 Lmol�cm�. The absorbance of the

coloured extracts obeyed the Beer's law in the range 0.01–2.1 mgL�. The extraction

of chromium was the highest during extraction from the sulfuric acid medium

(0.05– 3 M H2SO4). The method extended to the determination of various types of

soils and sewage doped with chromium(VI).

Abdollahi reported a simultaneous spectrophotometric determination of

chromium(VI) and iron(III) with mixed reagents by H-point standard addition method

[69]. Mixed reagents of diphenylcarbazide and 1,10-phenanthroline in a non-ionic

micellar solution of Triton X-100 was used as a selective chromogenic system for

determination of Cr(VI) and Fe(III). The presence of a micellar system allowed to

eliminate the previous solvent extraction step that was necessary for the determination

of slightly soluble metal complexes in the absence of micelles. This reduced the cost

and toxicity of the method. The total relative standard error for 15 synthetic samples

were in the range 0.20–6.00 ���� of Cr(VI) and 0.20–8.00 ���1 of Fe(III) were

1.5 %. The methods were applied to the determination of Cr and Fe in several

synthetic alloy solutions.

Melwanki and Seetharamappa reported propericiazine as a spectrophotometric

reagent for the determination of chromium in environmental samples [70].

Propericiazine formed a red colored radical cation, exhibited maximum absorption at

510 nm in H3PO4 medium. Beer's law was valid over the concentration range of

0.15- 2.25 mgL-1. The Sandell's sensitivity of the reaction was found to be

3.42 ngcm-2.

Mohamed and El-Shahat reported a spectrophotometric determination of

chromium and vanadium [71]. The method was based on the reactions with

perphenazine which instantaneously formed a red colored product and exhibited a

maximum absorbance at 526 nm.

135

Melwanki and Seetharamappa described a spectrophotometric determination

of chromium(VI) in soil and steel samples using methdilazine hydrochloride [72]. The

reagent formed a red colored species instantaneously on reaction with chromium(VI)

in phosphoric acid medium and exhibited maximum absorbance at 512 nm with

Sandells’s sensitivity of 3.1 ngcm-2. Beer's law was obeyed over the concentration

range of 0.1-1.8 ppm.

Llobat-Estelles et al. reported a method for the determination of chromium in

presence of V(V), Mo(VI) and iron(III) [73]. The effects of interferences were

evaluated by using the apparent content curves method and their separation was

performed by solid-phase extraction with an anionic exchanger. The sample treatment

conditions and the influence of the sample conductivity were studied. Tolerance limits

were established and the proposed procedure was used to determine chromium in

certified samples and for speciation of chromium in waste water.

Revanasiddappa and Kiran Kumar reported citrazinic acid as new coupling

agent for the indirect spectrophotometric determination of chromium by the oxidation

of hydroxylamine in acetate buffer of pH 4.0- 0.5 to nitrite [74]. Molar absorptivity

and Sandell's sensitivity of the reaction system were found to be 2.12×104 Lmol-1cm-1

��������",*��� �-2 at 470 nm. Beer's law was obeyed over the concentration range of

2-��������� ����� �&-.'���������������!�� ����������������� ������������/�������*�

hours.

Abdel-Aaty developed a simple and sensitive method for the

spectrophotometric determination of chromium(VI) based on its reaction with

chlorpromazine-HCl, which formed a red product [75]. Beer’s law was obeyed in the

range 0-��������-1 of Cr(VI) and the molar absorptivity value of the system was

3.28×104 Lmol-1cm-1.

Xin et al. reported chlorophosphonazo as a reagent for the spectrophotometric

determination of chromium in drinking water and alloy steel [76]. Proposed method

was based on the decolorization of chlorophosphonazo-pA in the presence of

perchlorate and molar absorptivity value of the system was 3.33×105 Lmol-1cm-1.

136

Revanasiddappa and Kiran Kumar used trifluoperazine hydrochloride as a

reagent for the rapid spectrophotometric determination of chromium [77]. The method

was based on the oxidation of trifluoperazine hydrochloride by Cr(VI) in the presence

of H3PO4. The red colored species exhibited an absorption maximum at 505 nm. The

system was obeyed Beer's law at 2-�0�������1�&-.'������������!�� �����������������

molar absorptivity of the color system was 2.08×104 Lmol-1cm-1 and the developed

color was stable for 2 hours. Leuco xylene cyanol-FF a sensitive reagent was used for

the selective spectrophotometric determination of trace amounts of chromium in

steels, industrial effluents and pharmaceutical samples [78]. The method was based on

the oxidation of leuco xylene cyanol-FF to its blue form of xylene cyanol-FF by

Cr(VI) in H2SO4 medium (pH 1.2-2.4), the absorbance of the formed dye was

measured in an acetate buffer medium (pH 3.0-4.6) at 615 nm. The method was

obeyed Beer's law in the concentration range of 0.05-��,�� ���-1 of chromium.

Molar absorptivity and Sandell’s sensitivity of the system was 8.23×104 Lmol-1cm-1

���������*���� �-2 respectively.

Melwanki and Seetharamappa used isothipendyl hydrochloride as a sensitive

and selective reagent for the simple spectrophotometric determination of

chromium(VI) [79]. The reagent reacted with chromium, which formed a red colored

species in H3PO4 medium, which exhibited maximum absorbance at 510 nm with

Sandell’s sensitivity of 2.28 ngcm-2. Beer's law was valid over the concentration range

of 0.1-1.9 mgL-1 of chromium.

Carvalho et al. reported 4-(2-thiazolylazo)-resorcinol (TAR) as a reagent for

the spectrophotometric determination of chromium [80]. Chromium reacted with

TAR, which formed a red complex at pH 5.7. Beer’s law was obeyed in the

concentration range 0.05-�������-1 of Cr(VI) and molar absorptivity of the system

was 2.72×104 Lmol-1cm-1 at 545 nm. The selectivity was improved by using EDTA

and citrate as masking agents.

Stoyanova reported a catalytic spectrophotometric method for the

determination of chromium(VI) [81]. The method was based on the catalytic effect of

chromium(VI) on the oxidation of sulphanilic acid (SA) by hydrogen peroxide in the

presence of p-aminobenzoic acid (PABA) as an activator. The reaction was followed

137

spectrophotometrically by tracing the formation of the reaction product at 360 nm

after 15 minutes of mixing the reagents. The optimum reaction conditions are

4.0×10-3 M sulphanilic acid, 0.57 M H2O2 , 1×10-3 M p-aminobenzoic acid and 0.04

M acetic acid - boric acid - orthophosphoric acid buffer solution (pH 6.6) at 50 °C.

The linear range of the calibration graph was up to 140 ngmL-1 and the detection limit

was 10 ngmL-1. Interferences of Cu(II) and Cr(III) ions were masked. The method

was applied to the analysis of Cr(VI) in industrial water with recoveries of 95.2–

104.5% and RSD of 2.9–5.8%.

Narayana and Cherian described a rapid spectrophotometric determination of

trace amounts of chromium using variamine blue as a chromogenic reagent [82]. The

method was based on the reaction of chromium(VI) with potassium iodide in acid

medium to liberate iodine. The liberated iodine which oxidized variamine blue to

violet colored species with an absorption maximum 556 nm. Beer’s law was obeyed

in the range 2-12 µgmL-1 of chromium(VI). The molar absorptivity, Sandell’s

sensitivity, detection limit and quantitation limit of the method were found to be

0.911×104 Lmol-1cm-1, 1.14×10-2 µgcm-2, 0.02 µgmL-1 and 0.07 µgmL-1

respectively. The chromium(III) was determined after its oxidation with bromine

water in alkaline medium to chromium(VI).

Stoyanova developed a catalytic spectrophotometric determination of

chromium [83]. The method was based on the catalytic effect of chromium(III) and

chromium(VI) on the oxidation of sulfanilic acid by hydrogen peroxide. The reaction

was followed spectrophotometrically by measuring the absorbance of the reaction

product at 360 nm. Two calibration graphs (for chromium(III) up to 100 ngmL-1, and

for chromium(VI) up to 200 ngmL-1) were obtained using the fixed time method with

detection limits of 4.9 ngmL-1 and 3.8 ngmL-1 respectively.

Zaitoun described a spectrophotometric determination of chromium(VI) using

cyclam (1,4,8,11-tetraazacyclotetradecane) as a reagent [84]. This method was based

on the absorbance of its complex with cyclam and the complex showed a molar

absorbtivity of 1.5×104 Lmol-1cm-1 at 379 nm. Under optimum experimental

conditions at a pH of 4.5 and 1.960×103 mgL-1 cyclam were selected, and all

measurements were performed 10 minutes after mixing. Major cations and anions did

138

not show any interference. Beer’s law was applicable in the concentration range

0.2–20 mgL-1 with a detection limit of 0.001mgL-1. The standard deviation in the

determination was ± 0.5 mgL-1 for a 15.0 mgL-1 solution (n=7). The described method

provides a simple and reliable for the determination of chromium in real samples.

Cherian and Narayana reported a spectrophotometric determination of

chromium(VI) using saccharin as a reagent [85]. In this method chromium(VI)

oxidized hydroxylamine in acetate buffer of pH 4.0 to nitrite, which then diazotized

p-nitroaniline or sulphanilamide to a diazonium salt. These diazonium salts were then

coupled with saccharin in an alkaline medium, which formed azo dyes with

absorption maximum at 372 and 390 nm for p-nitroaniline and sulphanilamide

respectively. The method obeyed Beer’s law in the concentration range of 1-16

µgmL-1 for chromium with p-nitroaniline-saccharin and 0.6-14 µgmL-1 for chromium

with sulphanilamide-saccharin couples. The molar absorptivity and Sandel’s

sensitivity of the systems with p-nitroaniline-saccharin and sulphanilamide-saccharin

couples were found to be to be 5.41×103 Lmol-1cm-1 and 1.93×10-3 µgcm-2 and

2.63×104 Lmol-1cm-1 and 3.90×10-3 µgcm-2 respectively.

Rao et al. described a spectrophotometric determination of chromium(III) after

extraction of its n-methylaniline carbodithioate complex into molten naphthalene [86].

Maximum extraction was obtained in the pH range of 2.0–3.5. Naphthalene

containing the metal complex was dissolved in DMF and the resulting solution

obeyed Beer’s law at 340 nm in the concentration range of 2.6–31."������� ����� ��

in 10 mL of the final solution. The molar absorptivity and Sandell’s sensitivity are

8.2×103 Lmol-1cm-1���������*,��� �-2 respectively. Interference of other metal ions

was studied and the method can be used for the determination of chromium in alloys

and industrial effluents.

Fabiyi and Donnio used variamine blue as a chromogenic reagent for rapid

spectrophotometric determination of nano amount of chromium [87]. The proposed

method was based on the reaction of chromium(VI) with potassium lodide in acid

medium to liberate iodine. The liberated iodine that oxidized variamine blue to violet

colored substances exhibited an absorption maxmium at 615 nm. The molar

139

absorptivity and Sandel’s sensitivity were found to be 8.12×103 Lmol-1cm-1 and

2.36×10-3 µgcm-2, respectively. The determination of chromium was found to be

0.0003–15 mgmL-1. Chromium(III) was determined after its oxidation to Cr(lV) with

bromine water in alkaline medium. The optimum reaction conditions, other analytical

parameters and interference effect of several ions were reported. The reagents

mentioned above are reported to be carcinogenic, while few others are less selective

and are time consuming. The need for a sensitive and simple method for the

determination of chromium is therefore clearly recognized.

The aim of the present work is to provide a simple and sensitive method for

the determination of chromium using toluidine blue and safranine O as new reagents.

The proposed method has been employed to the determination of chromium in steel,

soil, natural water and pharmaceutical samples.

140

5.3 APPARATUS

A Systronics 2201 UV-VIS Double Beam Spectrophotometer with 1 cm

quartz cell was used. A WTW pH 330 pH meter was used.

5.4 REAGENTS AND SOLUTIONS

All chemicals were of analytical reagent grade or chemically pure grade and

distilled water was used throughout the study. Chromium(VI) stock solution (1000

μgmL-1) was prepared by dissolving 0.2830 g of K2Cr2O7 in 100 mL standard flask

with distilled water and standardized by titrimetric method [18]. The stock solution

was further diluted as needed. Chromium(III) stock solution (1000 μgmL-1) was

prepared by dissolving 0.2830 g of K2Cr2O7 in 50 mL of water, added 1 mL

saturated sodium sulfite solution, acidified with 1 mL 2.5 M sulfuric acid, and then

boil for 2 minutes to remove excess SO2 and diluted with water to 100 mL. Suitable

volume of this solution was diluted to obtain the working standard. The following

reagents were prepared by dissolving appropriate amounts of the reagents in distilled

water: toluidine blue (0.01 %), safranine O (0.02 %), potassium iodide (2 %), acetate

buffer (pH = 4), bromine water (saturated), sulfosalicylic acid (5 %), potassium

hydroxide (4.0 M), sulfuric acid (2.5 M) and hydrochloric acid (2.0 M).

5.5 PROCEDURES

5.5.1 Using Toluidine Blue as a Reagent

5.5.1.1 Determination of chromium(VI)

Aliquots of sample solution containing 0.5-12.4 μgmL-1 of chromium(VI)

were transferred into a series of 10 mL calibrated flasks. Potassium iodide (2 %,

1 mL) and hydrochloric acid (2 M, 1 mL) were added and mixture was gently shaken

until the appearance of yellow color indicating the liberation of iodine. Toluidine blue

(0.01 %, 0.5 mL) was then added and the reaction mixture was shaken for 2 minutes

for maintaining pH = 4, 2 mL of acetate buffer was added. The contents were diluted

to 10 mL with distilled water and mixed well. The absorbance of the resulting

solutions were measured at 628.5 nm against reagent blank. Reagent blank was

141

prepared by replacing the analyte (chromium) solution with distilled water. The

absorbance corresponding to the bleached color, which in turn corresponds to the

analyte (chromium) concentration was obtained by subtracting the absorbance of the

blank solution from that of the test solution. The amount of the chromium present in

the volume taken was computed from the calibration graph (Figure VA1).

5.5.1.2 Determination of chromium(III)

Aliquots of sample solution containing 0.5-12.4 μgmL-1 of chromium(III)

were transferred in to a series of 10 mL calibrated flasks. A volume of 0.5 mL

saturated bromine water and 0.5 mL of 4 M KOH solution were added to each flask

and allowed to stand for 5 minutes. Then 0.5 mL of 2.5 M sulfuric acid and 0.5 mL of

5 % sulfosalicylic were added and then above procedure for chromium(VI) was

followed. The absorbance of the resulting solution was measured at 628.5 nm against

reagent blank.

5.5.2 Using Safranine O as a Reagent

5.5.2.1 Determination of chromium(VI)

Aliquots of sample solution containing 0.4-13.8 μgmL-1 of chromium(VI)

were transferred into a series of 10 mL calibrated flasks. Potassium iodide (2 %,1 mL)

and hydrochloric acid (2 M, 1 mL) were added and mixture was gently shaken until

the appearance of yellow color indicating the liberation of iodine. Safranine O (0.02

%, 0.5 mL) was then added and the reaction mixture was shaken for 2 minutes, for

maintaining pH = 4, 2 mL of acetate buffer was added. The contents were diluted to

10 mL with distilled water and mixed well. The absorbance of the resulting solutions

were measured at 532 nm against reagent blank. Reagent blank was prepared by

replacing the analyte (chromium) solution with distilled water. The absorbance

corresponding to the bleached color, which in turn corresponds to the analyte

(chromium) concentration, was obtained by subtracting the absorbance of the blank

solution from that of the test solution. The amount of the chromium present in the

volume taken was computed from the calibration graph (Figure VA2).

142

5.5.2.2 Determination of chromium(III)

Aliquots of a sample solution containing 0.4-13.8 μgmL-1 of chromium(III)

was transferred in to a series of 10 mL calibrated flasks. A volume of 0.5 mL

saturated bromine water and 0.5 mL of 4 M KOH solution were added to each flask

and allowed to stand for 5 minutes. Then 0.5 mL of 2.5 M sulfuric acid and 0.5 mL of

5 % sulfosalicylic were added and then above procedure for chromium(VI) was

followed. The absorbance of the resulting solution was measured at 532 nm against

reagent blank.

5.5.3 Analysis of Chromium SteelSteel (0.05 g per 100 mL) was dissolved in approximately 8 mL of aqua regia.

It was evaporated nearly to dryness on a sand bath, sulfuric acid (1-2 mL, 1:1 ) was

added and evaporated until salts crystallized, to this 10 mL of water was added. The

solution was warmed, filtered. The interference of vanadium(V) can be overcome by

extraction of chromium(VI) as chromyl chloride in 5 mL of methyl isobutyl ketone

(MIBK) after the addition of 5 mL of 5 M HCl to provide an overall acidity of

0.3-0.5 M [88]. chromium(VI) in organic layer was stripped by equilibration with 5

mL of water for determination. Suitable aliquots of sample solutions were analyzed

according to the procedure for chromium(III).

5.5.4 Determination of Chromium in Natural Water Samples

Each filtered environmental water samples (100 mL) were analyzed for

chromium. They tested negative. To these samples known amounts (not more than

����� ���-1) of chromium(VI) were spiked and analyzed for chromium by the

proposed procedure. Solutions were also analyzed according to the standard

diphenylcarbazide method [89].

5.5.5 Determination of Chromium in Soil Samples

A known amount of (1 g) air dried soil samples, spiked with known amounts

of chromium(VI) was taken and then fused with 5 g anhydrous sodium carbonate in a

silica crucible and evaporated to dryness after the addition of 25 mL of water. The

dried material was dissolved in water, filtered through whatman No. 40 filter paper in

to 25 mL calibrated flask and neutralized with dilute ammonia. It was then diluted to

143

a known volume with water. An aliquot of this sample solution was analyzed for

chromium(VI). Solutions were also analyzed according to the standard

diphenylcarbazide method [89].

5.5.6 Analysis of Pharmaceutical Samples

Samples of the finely ground multivitamin–multimineral tablets containing

chromium(III) were treated with 5 mL of nitric acid and the mixtures were evaporated

to dryness. The residue was leached with 5 mL of 0.5 M H2SO4. The solution was

diluted to a known volume with water. Suitable aliquots of the sample solution were

analyzed according to the procedure for chromium(III).

5.6 RESULTS AND DISCUSSION

5.6.1 Absorption Spectra

5.6.1.1 Using toluidine blue as a reagent

This method is based on the reaction of chromium(VI) with potassium iodide

in acid medium to liberate iodine. This liberated iodine bleaches the blue color of the

toluidine blue. The decrease in absorbance at 628.5 nm is directly proportional to the

chromium(VI) concentration. The absorption spectra of the colored species of

toluidine blue is presented in Figure VA and reaction system is also presented in

Scheme V.

5.6.1.2 Using safranine O as a reagent

Similarly this method is also based on the reaction of chromium(VI) with

potassium iodide in acid medium to liberate iodine. This liberated iodine bleaches the

pinkish red color of the safranine O. The decrease in absorbance at 532 nm is directly

proportional to the chromium(VI) concentration. The absorption spectra of the

colored species of safranine O is presented in Figure VA and reaction system is also

presented in Scheme V.

5.6.2 Effect of Iodide Concentration and Acidity

The oxidation of iodide to iodine is effective in the pH range 1.0 to 1.5, which

can be maintained by adding 1 mL of 2 M HCl in a final volume of 10 mL. The

liberation of iodine from KI in an acid medium is quantitative. The appearance of

144

yellow color indicates the liberation of iodine. Although any excess of iodine in the

solution will not interfere. It is found that 1 mL each of 2 % KI and 2 M HCl are

sufficient for the liberation of iodine from iodide by chromium(VI) and 0.5 mL of

0.01 % toluidine blue and 0.5 mL of 0.02 % safranine O are sufficient for the

decolorization reaction. The bleached reaction system is found to be stable for about

4 hours for each toluidine blue and safranine O reagents.

Constant and maximum absorbance values are obtained in the pH=4±0.2.

Hence the pH of the reaction system was maintained at 4±0.2 throughout the study.

This could be achieved by the addition of 2 mL of 1 M sodium acetate solution in a

total volume of 10 mL.

5.6.3 Analytical Data

5.6.3.1 Using touidine blue as a reagent

Adherence to Beer’s law is studied by measuring the absorbance values of

solutions varying chromium concentration. A straight line graph is obtained by

plotting absorbance against concentration of chromium. Beer’s law is obeyed in the

concentration range of 0.5-12.4 μgmL-1 of chromium (Figure VA1). The molar

absorptivity and Sandell’s sensitivity of system is found to be 1.457×104 Lmol-1cm-1

and 5.141×10-3 �� �-2 respectively. The detection limit (DL=3.3σ/S) and quantitation

limit (QL=10σ/S) (where σ is the standard deviation of the reagent blank (n=5) and S

is the slope of the calibration curve) of the chromium determination are found to be

����*����-1 �������,0����-1 respectively.

5.6.3.2 Using safranine O as a reagentAdherence to Beer’s law is studied by measuring the absorbance values of

solutions varying chromium concentration. A straight line graph is obtained by

plotting absorbance against concentration of chromium. Beer’s law is obeyed in the

concentration range 0.4–13.8 μgmL-1 of chromium (Figure VA2). The molar

absorptivity and Sandell’s sensitivity of the system is found to be 1.093×104

Lmol-1cm-1 and 6.849×10-3 �� �-2 respectively. The detection limit (DL =3.3σ/S) and

quantitation limit (QL=10σ/S) (where σ is the standard deviation of the reagent blank

145

(n=5) and S is the slope of the calibration curve) of the chromium determination are

�� ������/����""#����-1 a�����*#,����-1 respectively.

5.6.4 Effect of Divers Ions

The effect of various ions at microgram levels on the determination of

chromium is examined. The tolerance limits of interfering species are established at

those concentrations that do not cause more than ±2.0 % error in absorbance values of

����� ���������2��� �� ����������&"����-1). The results are given in Table 5A1.

The reaction involving chromium with potassium iodide, various ions such as Cu2+,

V5+, Fe3+, iodate and periodate are interfered. However, the tolerance level of some of

these ions may be increased by the addition of 1 mL of 1 % EDTA solution. Fe3+ can

be masked using sodium fluoride solution.

5.7 APPLICATIONS

The proposed method is applied to the quantitative determination of traces of

chromium in different samples such as alloys, water, soil and pharmaceutical samples.

The results are summarized in Table 5A2, 5A3 and 5A4. Statistical analysis of the

results by t-tests show that, there is no significant difference in accuracy and precision

of the proposed and reference method [89]. The precision of the proposed method is

evaluated by replicate analysis of samples containing chromium at five different

concentrations.

5.8 CONCLUSIONS

1. The reagents provide a simple and sensitive methods for the spectrophotometric

determination of chromium.

2. The reagents have the advantage of high sensitivity and selectivity.

3. The developed methods does not involve any stringent reaction conditions and

offers the advantages of high color stability compared to the standard

diphenylcarbazide method (30 minutes). The color stability of the reaction system

is found to be stable for 4 hours for toluidine blue and safranine O methods.

4. Statistical analysis of the results by the t- tests show that, there is no significant

146

difference in accuracy and precision of the proposed and reference method.

5. The proposed methods can be used for the determination of traces of chromium in

alloys, water, soil and pharmaceutical samples. A comparison of the method

reported is made with earlier methods and is given in Table 5A5.

FIGURE VA

ABSORPTION SPECTRA OF COLORED SPECIES TOLUIDINE BLUE(b) AND

SAFRANINE O(a)

Wavelength (nm)

200 300 400 500 600 700 800 900

Abso

rban

ce

0.0

0.5

1.0

1.5

2.0

a

b

147

SCHEME V

SCHEME OF THE REACTIONS

Cr2O72 + 6 I + 14 H 2 Cr3 + 3 I2 + 7 H2O

N

S+

CH3

NH2(CH3)2N

NH

S

CH3

NH2(CH3)2N

I2 , H+

Toluidine Blue(Colored) Toluidine Blue(Colorless)

N

N+

CH3

NH2NH2

CH3 NH

N

CH3

NH2NH2

CH3 I2 , H+

Safranine O (Colored) Safranine O (Colorless)

148

FIGURE VA1ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF CHROMIUM

USING TOLUIDINE BLUE AS A REAGENT

Concentration of chromium (µgmL-1)

0 2 4 6 8 10 12 14 16 18

Abs

orba

nce

0 .0

0.5

1.0

1.5

2.0

2.5

3.0

FIGURE VA2ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF CHROMIUM

USING SAFRANINE O AS A REAGENT

Concentration of chromium (µgmL-1)

0 2 4 6 8 10 12 14 16 18

Abs

orba

nce

0.0

0.5

1.0

1.5

2.0

149

TABLE 5A1 EFFECT OF DIVERSE IONS ON THE DETERMINATION OF CHROMIUM(VI) (2 μgmL-1)

Foreign io������������� ��������&���-1'������3���������������������� ��������&���-1)

Toluidine blue Safranine O Toluidine blue Safranine O

* Fe3+ 10 10 *V5+ 10 10

Ni2+ 75 100 Zn2+ 500 250

* Cu2+ 10 10 MoO42- 750 700

Cd2+ 500 600 PO43- 1000 1200

Ba2+ 750 750 Oxalate 1000 750

Bi3+ 1000 1000 F- 750 1000

Mn2+ 500 500 Sulfate 1000 1000

Al3+ 500 400 Chloride 750 1000

Ca2+ 50 75 Tartarate 1250 1000

Co2+ 75 50 Nitrate 1250 1500

Acetate 1100 1200

* Masked with masking agents.

150

TABLE 5A2DETERMINATION OF CHROMIUM IN ALLOY STEELS USING TOLUIDINE

BLUE AND SAFRANINE O REAGENTS

Toluidine blue Safranine O

Samples Cr Cr Recovery t- Cr Recovery t- certified found* (%) testd found* (%) testd

(%) (%) ± SD (%) ± SD

GKW Steel, 1.02 1.01± 0.015 99.02 0.37 1.00± 0.03 98.03 1.90India (0.0501g/100 mL);C 0.54, Mn 0.89, S 0.018,P 0.308, Si 0.33, V 0.13a

Stainless steel 18.00 17.96 ± 0.02 99.78 1.25 17.97 ± 0.05 99. 38 1.50 no.394(0.0503g/100 mL);Ni 8.12,Fe 70-71b

Ferrochrome, Fe 35c 65.00 64.78 ± 0.05 99.70 2.57 64.86 ± 0.04 99.78 1.40

*. Mean ± standard deviation( n=5)

a. Diluted to 5 times before analysis;

b. Diluted to 10 times before analysis;

c. Diluted to 15 times before analysis;

d. Tabulated t-value for 8 degree of freedom at P(0.95) is 2.306 .

151

TABLE 5A3DETERMINATION OF CHROMIUM IN SOIL SAMPLES AND NATURAL

WATER SAMPLES USING TOLUIDINE BLUE AND SAFRANINE O

REAGENTS

Toluidine blue Safranine O

Samples Cr(VI) Cr(VI) Relative Recovery t- Cr(VI) Relative Recovery t- added founda error (%) testb founda error (%) testb

�������������������-1 ���-1±�45����&$'�������������������������������������-1± SD (%)

Soil 4.0 4.02 ± 0.02 0.25 100.5 1.67 3.99 ± 0.04 -0.25 99.75 1.57

Samples 6.0 6.01 ± 0.01 0.17 100.2 0.36 6.02 ± 0.08 0.33 100.3 0.89

8.0 7.96 ± 0.02 -0.50 99.5 ----- ------- --- ----- ----

Natural 4.0 4.01 ± 0.02 0.49 100.2 0.83 4.01 ± 0.02 0.25 100.2 1.12

water 6.0 6.01 ± 0.01 0.17 100.1 2.00 5.99 ± 0.02 -0.17 99.83 1.11

Samples 8.0 8.02 ± 0.03 0.25 100.2 ---- ------- ---- ----- ----

a. Mean ± standard deviation(n=5)

b. Tabulated t-value for 8 degree of freedom at P(0.95) is 2.306 .

152

TABLE 5A4DETERMINATION OF CHROMIUM IN PHARMACEUTICALS SAMPLES

USING TOLUIDINE BLUE OR SAFRANINE O REAGENTS

Toluidine blue Safranine O

Samples Cr Cr Recovery Cr Recovery certified found* (%) found* (%) (mg/tablet) (mg/tablet ± SD) (mg/tablet ± SD)

Chromoplexa 0.200 0.199±0.010 99.5 0.196±0.040 98.0 (0.550 g/100 mL)

Fourts Bb 0.150 0.149±0.007 99.3 0.147±0.020 98.0 (0.650 g/100 mL)

Optisulinc 0.500 0.499±0.006 99.8 0.494±0.035 98.8 (0.300 g/100 mL)

a. Chromoplex (Aristo pharmaceuticals Ltd., Chennai-600 096, India)

Composition – Zinc sulphate monohydrate-27.50 mg; vitamin B1-10 mg; vitamin

B12-15mg; nicotinamide-50 mg; calcium pantothenate-12 mg; folic acid-1 mg;

vitamin C-150 mg;(0.550 mg).

b. Fourts B (Fourts India Laboratories Private Ltd., Kelambakkam-603 103, Tamil

Nadu, India)

Composition –Thiamine mononitrate-10 mg; riboflavin-10 mg; pyridoxine

hydrochloride-3mg; vitamin C-150 mg; zinc sulphate-80 mg; selenium-100 mg;

(0.650).

c. Optisulin (Dr. Reddy’s Laboratories Ltd., Hyderabad-500 016, India)

Composition – Zinc sulphate monohydrate-27.50 mg; vitamin B6-3 mg; vitamin

B12-15 mg; folic acid-1 mg; (0.3 g).

*. Mean ± standard deviation( n=5)

153

TABLE 5A4COMPARISON OF THE METHOD REPORTED WITH EARLIER METHODS

Reagent Method Beer’s law&���-1)

ε (Lmol-1cm-1)���&�� �-2)

λmax(nm)

Ref. No.

4-(2-Thiazolylazo)-resorcinol

Spectrophotometry 0.05-3.0 ε = 2.72×104

--------545 80

Variamine blue Spectrophotometry 2-12 ε = 0.911×104

ss = 1.14×10-2556 82

Cyclam Spectrophotometry 0.2-20 ε = 1.5×104

--------379 84

p-Nitroaniline-saccharin

Sulphanilamide-saccharin

Spectrophotometry 1-16

0.6-14

ε = 5.41×103

ss = 1.93×10-3

ε = 2.63×104

ss = 3.90×10-3

372

390

85

Variamine blue Spectrophotometry 0.0003-15 mgmL-1

ε = 8.12×103

ss = 2.36×10-3615 87

Proposed MethodToluidine blue

Safranine O

Spectrophotometry 0.5-12.4

0.4-13.8

ε = 1.457×104

ss = 5.141×10-3

ε = 1.093×104

ss = 6.849×10-3

628.5

532

ε = Molar absorptivity, ss = Sandell’s sensitivity

154

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