2. methods for spectrophotopmeric determination of vitamin...

33

2. METHODS FOR SPECTROPHOTOPMERIC

DETERMINATION OF VITAMIN C

Page No.

2.1 Use of Iron(III)-3-Hydroxy-2-aryl-4H-chromen-4-one complex 34

in the Spectrophotometric determination of vitamin C.

2.2 Spectrophotometric determination of vitamin C using 3-Hydroxy- 48

2-(3-methyl thiophen-2-yl)-4H-chromen-4-one as a reagent.

2.3 Spectrophotometric determination of vitamin C in pharmaceuticals using 62

Iron(III)-6-Chloro-3-hydroxy-2(4-methoxyphenyl)-4H-chromen-4-one

complex.

2.4 Spectrophotometric determination of vitamin C using Fe(II)-5-Chloro-7- 76

iodo-8-hydroxyquinoline complex.

2.5 Use of calmagite in the determination of vitamin C involving 90

Ce(IV)-Ce(III) redox couple.

2.6 Spectrophotometric determination of vitamin C using Iron(II)-Batho- 104

phenanthroline complex.

34

2.1 Use of Iron(III)-3-Hydroxy-2-aryl-4H-chromen-4-one complex

in the Spectrophotometric determination of vitamin C

During the last few decades, many methods based on iron(III)-iron(II) system

have been proposed for the assay of ascorbic acid samples of diverse nature. These

procedures involve the reagents such as 2,2‟-bipyridyl229-237

, 1-10-phenanthroline238-

248, ferrozine

254-256, 2-(5-bromo-2-pyridylazo)-5-dimethylaminophenol

265, TPTZ

258-260

and nitroso-R-salt262

etc. Of them, the methods involving 2-2‟-bipyridyl and 1,10-

phenanthroline find extensive use in the development in the analysis of vitamin C.

Many of these methods are time consuming as full colour development takes 30-60

min. Recently simplification of the procedure involving 3-hydroxy-2-(2-thienyl)-4H-

chromen-4-one complex has been reported from our laboratory which requires only

one min waiting time instead of 30 or 60 min. Many methods using 1,10-

phenanthroline need either background correction as done by Cu-catalysed oxidation

or the addition of NH4F as the inhibitor of the light reduction of iron(III)-O-phen

complex. An improvement has been made using the orange- red iron(II)-O-phen

chelate in aqueous miscellar medium formed in presence of the cationic surfactant

cetylpyridinium bromide. The methods using ferrozine or 2-OCHT suffer

interferences from metal ions such as Co(II), Ni(II) ,Cu(II), Pd(II) and iron (II) in

addition to the interferences caused by tartaric acid, citric acid, oxalic acid, riboflavin

and oxidants.

3-Hydroxy-2-aryl-4H-chromen-4-one (HAC) has been found to be an

appropriate reagent that forms a colored complex with iron (III) in almost neutral

solutions. The complex gets extracted into chloroform to give a reddish brown extract.

The proportionate decrease in absorbance of the complex with the addition of

increasing amount of ascorbic acid forms the basis of the proposed method. The

method based on the extraction of iron (III) –HAC complex provides the desirable

features of simplicity and rapidly besides having better sensitivity and selectivity. The

detailed studies pertaining to the proposed method are presented here.

35

2.1.1 Experimental

Instrument

A Hitachi schimadzu spectrophotometer (model UV-140-02) with a pair of

matched 1cm quartz cells was used for absorbance measurements.

Reagents and Solutions

Iron (III) solution

A (1mg ml-1

) iron (III) solution was prepared by dissolving accurately weighed

amount of Ammonium ferric sulphate in 100 ml of deionised water containing 0.5 ml

of concentrated sulphuric acid. A lower concentration (10 μg ml-1

) was obtained by

dilution of the stock solution.

3-Hydroxy-2-aryl-4H-chromen-4-one (HAC) solution

A 0.05% (w/v) solution was obtained by dissolving the reagent in ethanol.

O

O

OH

3-Hydroxy-2-aryl-4H-chromen-4-one (HAC)

Ascorbic acid solution

A fresh aqueous solution of ascorbic acid (100 μg ml-1

) was used. A lower

concentration (10 μg ml-1

) was obtained by dilution of the stock solution.

Deionized water was used for preparing solutions and all the reagents used were

of analytical grade unless otherwise stated.

36

Procedure

Into a 100 ml separatory funnel, 10 μg of iron (III) solution was pipetted

followed by the addition of an aliquot of ascorbic acid and 0.5 ml of HAC solution.

Enough water was added to make the aqueous phase to 10 ml. The resulting complex

was extracted for 0.5 min with 10 ml of chloroform. The coloured extract was taken

into a 10 ml volumetric flask and the volume was made up to the mark with

chloroform, if required. The absorbance of the reddish brown complex was measured

at 405 nm against the reagent blank prepared similarly and the vitamin C content is

determined from the calibration curve constructed under the optimum conditions of

the procedure.

Analysis of tablets /capsules

The tablets or capsules (5-7 items) were crushed to the powder form and a

known weight equivalent to 100 mg of ascorbic acid was dissolved in deionized water.

The solution was filtered into a 100 ml volumetric flask and made up to the volume. A

lower concentration (100 μg ml-1

) was obtained by suitable dilution of this solution.

The diluted solution was analysed by the proposed procedure.

2.1.2 Results and discussion

Spectral characteristics

The electronic spectrum of the iron (III)-HAC complex was studied over the

range 375-720 nm which shows one absorption band in the region 405-407 nm (Fig.

01). The complex absorbs strongly at 405 nm, where minimum absorption was shown

by the blank. Therefore, absorbance measurements were made at 405 nm.

37

Fig. 01 Absorption spectrum of Iron(III)-HAC complex

(Conditions: Iron(III) =10 μg; HAC solution = 0.5 ml )

A – Reagent blank against chloroform

B – Complex against reagent blank

350 400 450 500 550 600 650 700 750

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

B

A

Absorb

ance

Wavelength (nm)

38

Choice of solvent

The extraction behaviour of the complex in different solvents viz.

Dichloromethane, benzene, chloroform, carbontetrachloride, xylene, n-hexane, iso-

Amyl acetate and iso-Propyl alcohol was studied as shown in Table 3. Among the

solvents tested, dichloromethane, benzene, chloroform, carbon tetrachloride,

xylene were found to extract the complex but little extraction was observed into iso-

amyl acetate and iso-propyl alcohol. Chloroform was chosen as an extractant because

it gave the highest absorbance.

Table 3

Extraction Behaviour of the complex in Different solvents

Solvent Absorbance*

Chloroform 0.73

Dichloromethane 0.70

Carbon tetrachloride 0.69

Benzene 0.65

Xylene 0.39

n-Hexane 0.17

iso-Amyl acetate 0.00

iso-Propyl alcohol 0.00

* Measured against respective blank

Effects of reaction variables

The various parameters which can influence the extraction and absorbance of

the complex as well were studied. These include the changes in concentration of the

reagent, pH, equilibration time and temperature. For such studies, the aqueous solution

39

Fig. 02 A - Effect of HAC concentration

B - Effect of pH

0.0 0.5 1.0 1.5 2.0

0.2

0.3

0.4

0.5

0.6

0.7

0.80 2 4 6 8 10

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

B

A

B

Ab

so

rba

nce

Ab

so

rba

nce

HAC Concentration (ml)

A

pH

40

Fig.03 Effect of equilibration time

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0.60

0.62

0.64

0.66

0.68

0.70

0.72

0.74

0.76

0.78

0.80

Ab

so

rba

nce

Equilibration Time (min)

41

containing, 10 μg of Iron (III) and 0.5 ml of the reagent (HAC) solution was

equilibrated with an equal volume of chloroform for 0.5 min. The other conditions

used are shown at the bottom of Table 4.

Effect of HAC concentration

An increase in the HAC concentration up to 0.4 ml of the reagent solution

enhances the absorbance of the complex which thereafter remains the same up to 1.5

ml but there is a gradual decrease in absorbance above this concentration (Table 4,

Fig.02 Curve A). Hence 0.5 ml of the HAC solution was used for further studies.

Effect of pH

The extraction of the Fe(III)-HAC complex was studied over a pH range 1.5-

10.2. It was observed that the complex gives maximum absorbance within range of pH

3.5-8.5 (Table 4, Fig. 02, Curve B). However, a decrease in absorbance is observed on

going to either side of the range.

Table 4

Optimization of reaction variables

HAC conc. (in ml) 0.1 0.2 0.4-1.0 2.0

Absorbance 0.26 0.53 0.73 0.70

pH 1.5 2.0 3.5-8.5 9.0 10.2

Absorbance 0.43 0.63 0.73 0.67 0.63

Equilibration time

(in min)

0.25 0.5-1.0 2.0

Absorbance 0.70 0.73 0.71

Temperature (oC) 20-35 40 45 50

Absorbance 0.73 0.70 0.68 0.67

Conditions: Iron(III) = 10 μg ; volume of reagent (HAC) solution = 0.5 ml ;

equilibration time = 0.5 min ; volume of aqueous phase = 10 ml ; volume of

chloroform = 10 ml; λ max = 405 nm.

42

Effect of the equilibration time

An increase in the time contact between two phases up to 0.5 min enhances the

extraction as seen by the corresponding by the increase in absorbance of the complex.

It remains constant up to 1 min of equilibration time (Table 4, Fig.03). Thereafter, a

slight decrease in absorbance is observed with the equilibration time of 2 min or more.

Therefore, equilibration time of 0.5 min was chosen.

Effect of temperature

The effect of temperature of aqueous solution was found to have little effect on

the absorbance of the complex over the range 20-350C (Table 4). Further increase in

temperature of the aqueous phase causes a gradual decrease in the absorbance of the

complex.

Beer’s law

Under the optimum conditions, a standard calibration curve was constructed at

405 nm by adding different amount of ascorbic acid to iron(III) solution. A linear

relationship between absorbance and concentration of the analyte was observed up to

2.4 μg ml-1

of ascorbic acid (Table 5, Fig. 04).

Table 5

Absorbance Values at Different Concentration of Ascorbic Acid

Amount of ascorbic acid (µg/ 10ml) Absorbance

0 0.73

2 0.67

6 0.57

8 0.48

10 0.45

12 0.38

16 0.27

20 0.15

24 0.04

43

Fig. 04 Beer‟s law curve for varied amount of ascorbic acid

0 5 10 15 20 25

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Ab

so

rban

ce

Conc. of Ascorbic Acid ( g/ 10ml)

44

Interference studies

The effect of possible ingredients likely to be present in pharmaceutical

products was studied. The result of such studies is shown in Table 6, where the

different substances are classified under the headings sugars, vitamins and amino

acids, organic acids, cations and anions etc. Sugars are tolerated in good amounts

Table 6

Effect of diverse substances

Substance added# Tolerance Limit (mg per 10ml)

Sugars

Glucose, Fructose 200

Maltose 150

Sucrose 100

Lactose 50

Vitamins & Amino Acids

Thiamine hydrochloride 5.0

Methionine, Asparatic acid 5.0

Nicotinic acid 5.0

Pyridoxine hydrochloride 4.0

Glutamic acid 1.0

Nicotinamide 0.6

Riboflavin, Cyanocobalamin 0.1

Folic acid 0.05

Cysteine 0.03

Organic acids

Benzoic acid 10

Maleic acid 7.0

Succinic acid 2.0

Contd.

45

Tartaric acid, Salicylic acid 0.5

Citric acid 0.05

Cations and Anions

Ca(II), Mg(II) 5.0

Zn(II) 0.8

Al(III) 0.1

Co(II), Mn (II) 0.03

Cl-, SO4

2- 20

F-

2.0

NO3- 1.0

Miscellaneous

Acetone 200

Formaldehyde 100

Thiourea, Urea 50

Glycerol 40

# Substances were added prior to the addition of ascorbic acid.

at the levels indicated but the tolerance limit for the studied vitamins and amino acids

is relatively low especially with riboflavin, cyanocobalamin, cysteine and folic acid.

Tested organic acids other than citric acid are tolerated to a varying degree. Among

the cations and anions Co(II) and Mn(II) are tolerated in traces whereas Al(III)

interfere with the determination. Some other substances as mentioned under the

heading „miscellaneous‟ are also tolerated.

46

Applications

The worked out method for the determination of ascorbic acid is fast and

facile. The method is quite useful and allows satisfactory determination of ascorbic

acid in the presence of common ingredients in pharmaceutical formulations (Table 7).

Table 7

Analysis of pharmaceutical products

Sr.No. Preparation Ascorbic Acid Content per tablet (in mg)

Claimed Found

1 Celin 500 498.5

2 Limcee 100 98.7

3 Supradyn (multivit.) 150 145.8

4 Celin( Chewable) 200 198.2

47

2.1.3 Abstract

A rapid spectrophotometric method for

the determination of ascorbic acid is described.

It involves the reduction of iron(III) to iron(II)

with ascorbic acid and the formation of brown

coloured complex by the reaction of iron(III)

with 3-Hydroxy-2-aryl-4H-chromen-4-one,

followed by the extraction of the complex into

chloroform and measuring the absorbance at

405 nm. The Beer’s law is obeyed up to 2.4 µg ml-

1 of ascorbic acid having molar absorptivity and

Sandell’s sensitivity of 5.897 x 105 l mol-1 cm-1 and

2.986 x 10-4 µg cm-2 respectively. The interference

of various substances commonly added to

pharmaceuticals were studied. The procedure

has been applied to the assay of pharmaceutical

preparations containing vitamin C.

48

2.2 Spectrophotometric determination of vitamin C using 3-

Hydroxy-2-(3-methyl thiophen-2-yl)-4H-chromen-4-one as a

reagent

Ascorbic acid commonly known as vitamin C, is an important water soluble

vitamin. Humans and apes cannot synthesize ascorbic acid due to lack of

gulonolactone oxidase enzyme and hence ascorbic acid has to be supplemented from

external sources, mainly through vegetables, fruits and pharmaceutical products.

Vitamin C is one of the most essential vitamin for both pharmaceutical and food

processing industries in view of its nutritional significance, varied use in food products

and its daily dose requirement for optimum health.

A large number of methods for determination of ascorbic acid include

titrimetry22-29

, voltametry116-120

, amperometry128-132

, potentiometry136-141

,

chemilumescence69-81

and flow injection154, 227, 228

analysis. These methods have been

used to increase the analytical sensitivity for ascorbic acid and some of them are

automated, but specialized equipments are required for these procedures. Besides,

spectrophotometric methods are commonly used for the determination of ascorbic

acid. Many reagents such as; fast red186,187

, leucomalachite green189

, rhodamine B90-92

and methyl viologen188

etc.to mention a few, are used in the determination of ascorbic

acid.

A simple and sensitive analytical method is required for the analysis of a

variety of samples. The method using 3-hydroxy-2-(3-methyl thiophen-2-yl)-4H-

chromen-4-one (HMTC) is described which meets such requisite characteristics of a

photometric method. The proposed method is based on the proportionate decrease in

the colour intensity of iron (III)-HMTC complex by the addition of ascorbic acid

followed by its extraction into chloroform.

49

2.2.1 Experimental

Instrument

A Hitachi schimadzu spectrophotometer (model UV-140-02) with a pair of

matched 1cm quartz cells was used for absorbance measurements.

Reagents and solutions

The reagents used were of analytical grade and redistilled water was used for

the preparation the solutions.

Iron (III) solution

A (1mg ml-1




) was obtained by


3-Hydroxy-2-(3-methyl thiophen-2-yl)-4H-Chromen-4-one (HMTC) solution


O

O

S

H3C

OH

3-Hydroxy-2-(3-methyl thiophen-2-yl)-4H-Chromen-4-one

50



) was used. A lower



Procedure

In a 100 ml seperatory funnel, 10 μg of iron (III) solution was taken followed

by addition of an aliquot of ascorbic acid and 0.5 ml of HMTC solution. Enough water

was added to make the aqueous phase to 10 ml. The brown colored complex so

formed was extracted for one min with 10 ml of chloroform. The colored extract was

taken into a 10 ml volumetric flask and the volume was made up to the mark with

chloroform, if required. The absorbance of the reddish brown complex was measured

at 415 nm against the reagent blank prepared similarly. The contents of ascorbic acid

were calculated from the standard calibration curve prepared by taking different

concentration of ascorbic acid.


An accurately weighed amount of the powder, obtained by crushing 5-10

tablets and equivalent to 10 mg ascorbic acid was transferred to a 100 ml volumetric

flask. After dissolving the powder, the volume was made up to the mark with

deionized water. The working solution (10 μg ml-1

) was obtained by dilution of the

stock solution. An aliquot of this solution was analysed for ascorbic acid contents by

the recommended procedure.


Spectral Characteristics

During preliminary investigation it was observed that the color intensity of

iron(III)–HMTC complex diminishes with the increase in the amount of ascorbic acid.

This fact was exploited in the development of the proposed method. The complex gets

51

Fig.05 Absorption spectrum of Iron(III)-HMTC complex in chloroform.

(Conditions: Fe(III) =10 μg; HMTC solution = 0.5 ml )

A– Reagent blank against chloroform


400 450 500 550 600 650 700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

B

A

Ab

so

rban

ce

Wavelength (nm)

52

extracted into different solvents. The extraction of the brown colored complex thus

formed in acidic medium was carried out into different solvents such as chloroform,

dichloromethane, carbon tetrachloride, n-butanol, butan-2-one, n-hexane (Table 8). A

maximum absorbance was found in chloroform, which was preferred as the suitable

extractant for the complex. The absorption spectrum of the brown coloured complex

along with that of blank was studied. The spectrum shows an absorption band at 415-

418 nm (Fig. 05), where the reagent blank absorbs very little. Hence, all the

absorbance measurements were made at 415 nm.

Table 8

Extraction Behaviour of the complex in different solvents

Solvent Absorbance*

Chloroform 0.79



n-Butanol 0.38

Butan-2-one 0.34

n-Hexane 0.12



The effect of different variables affecting the absorbance and extraction of the

complex was studied (Table 9). In the study of these variables, 10 ml of the aqueous

phase solution containing 10 μg of iron(III) and varied amounts of the reagent and

ascorbic acid was equilibrated for one min with an equal volume of chloroform.

53

Effect of reagent concentration

The increase in the reagent (HMTC) concentration through 0.5 ml of HMTC

solution leads to increase in the absorbance which remains constant up to 1.0 ml of

HMTC solution (Table 9, Fig.06 Curve A). However, further increase in its

concentration causes a gradual decrease in the absorbance. Hence, 0.5 ml of the

HMTC solution was used for further studies.

Table 9


HMTC conc. (in ml) 0.2 0.4 0.5-1.0 1.5 2.0

Absorbance 0.62 0.77 0.79 0.77 0.75

pH 2.5 3.5-6.0 6.5 7.0 8.2 9.7 10.4

Absorbance 0.72 0.79 0.76 0.74 0.70 0.57 0.43

Equilibration time

(in sec)

15 25 50 60-

180

Absorbance 0.44 0.63 0.77 0.79

Temperature 20-

30

40 50

Absorbance 0.79 0.72 0.70

Conditions: Iron(III) (10 μg ml-1

) = 1ml , Volume of reagent (HTMC) = 0.5ml ,

Equilibration time = 1 min, Volume of aqueous phase = Volume of chloroform = 10

ml, λmax = 415 nm

54

Fig.06 A-Effect of HMTC concentration

B-Effect of pH

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0.60

0.62

0.64

0.66

0.68

0.70

0.72

0.74

0.76

0.78

0.80

0 2 4 6 8 10 12

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

BA

Absorb

ance

Absorb

ance

HMTC Conc. (ml)

B

A

pH

55

Fig.07 Effect of Equilibration Time

0 20 40 60 80 100 120 140 160 180 200

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

Ab

so

rba

nce

Equilibration Time (sec)

56

Effect of pH

The complex was studied over a pH range 2.5-10.4. It was observed that the

complex gives maximum absorbance in the acidic medium at the pH range 3.5-6.0

(Table 9, Fig.06 Curve B). However, a decrease is observed on going either side of

this range.

Effect of Equilibration Time

The effect of contact time over the range 15-180 sec was studied. On

equilibrating the iron (III) –HMTC complex with chloroform for 15 sec the

absorbance obtained was 0.44 which was increased to 0.77 in 50 sec and to a constant

value of 0.79 if the equilibration was increased to 1 min or beyond up to 3 min (Table

9, Fig.07). Therefore, 1 min equilibration time was chosen for the method.

Beer’s law and calibration curve

Using the optimum conditions of the procedure, a standard calibration curve

was constructed at 415 nm with the addition of different amounts of ascorbic acid to

iron(III) solution. A linear relationship between absorbance and the concentration of

the analyte was observed over the range 0.0-1.2 μg ml-1

(Table 10, Fig.08). The

calculated value of the molar absorptivity and Sandell,s sensitivity at 415 nm are 4.413

× 104 l mol

-1 cm

-1 and 0.00398 µg cm

-2 respectively.

Table 10

Absorbance Value at Different Concentration of Ascorbic Acid


0 0.79

1 0.76

2 0.72

4 0.65

6 0.57

8 0.49

10 0.42

12 0.36

13 0.34

14 0.32

57

Fig. 08 Beer‟s law curve for ascorbic acid

0 2 4 6 8 10 12 14 16

0.3

0.4

0.5

0.6

0.7

0.8

Absorb

ance

Concentration of ascorbic acid ( g/10ml)

58


Using the method, analysis of 10 μg of ascorbic acid in presence of some of

the commonly encountered substances such as sugars, vitamins and amino acids, was

carried out as shown in the Table 11.The tolerance limit forthese substances was

determined and for vitamins and amino acids it is comparatively good as compared to

cysteine, riboflavin and cyanocobalamin. Organic acids other than citric and salicylic

acid do not interfere. Among the tested cations and anions Al(III), Zn(II),Co(II) and

Mn(II) are tolerated in traces whereas thiosulfate seriously interferes with the

determination.

Table 11



Sugars

Sucrose 150


Lactose 50

Maltose 40


Glutamic acid 2.5


Methionine 1.0


Folic acid 0.25

Nicotinic acid 0.25

Nicotinamide 0.25

Riboflavin, cyanocobalamin 0.15

Cysteine 0.15

Asparaticacid 0.03

Contd.

59

Organic acids

Benzoic acid 20

Succinic acid 8.0

Maleic acid 3.0

Tartaric acid 1.5

Salicylic acid 0.4

Citric acid 0.1

Cations and Anions

Ca(II),Mg(II) 5.0

Zn(II), Al(III) 0.6

Co(II) , Mn (II) 0.03

Cl-, SO4

2- 80

F-

1.0

Miscellaneous

Formaldehyde 200

Glycerol 100

Thiourea, Urea 80

Starch 30


Applications

The method proposed for determination of ascorbic acid is quite simple and

sensitive. Most of the substances commonly found in pharmaceutical products were

found to be tolerated to a varying degree. The procedure is free from the various

interfering substances including sugars, amino acids and other additives. The method

is quite simple and rapid as it takes only 3-4 min for determination of ascorbic acid

60

after the preparation of sample solution. The method can be compared favourably with

the existing spectrophotometric methods. Some of the common pharmaceutical

preparations were successfully analysed by the proposed method as shown in the

Table 12.

Table 12


Sr. No. Preparation Ascorbic Acid Content per tablet (in mg)

Claimed Found

1 Celin 500 501.5

2 Limcee 100 100.5

3 Supradyn 150 149.5

4 Sym-o-vit 75 74.7

61

2.2.3 Abstract

The proposed method for ascorbic acid

determination is based on the extraction of a

Fe(III)- 3-hydroxy-2-(3-methyl thiophen-2-yl)-

chromen-4-one (HMTC) complex in acidic

medium (pH 3.5-6.0), where the absorbance of

the formed Fe(III)-HMTC chelate is measured at

415 nm after extraction with chloroform. The

absorbance of the complex gets decreased with

the increasing amount of ascorbic acid. The

proportionate decrease in absorbance forms the

basis of the proposed procedure and the molar

absorptivity and Sandell’s sensitivity for

vitamin C is found to be 4.413 × 104 l mol-1 cm-1

and 0.oo398 µg cm-2. Beer’s law is obeyed up to

1.2 μg ml-1 of ascorbic acid. The method has been

successfully applied to determination of ascorbic

acid in the pure state, dosage forms and

multivitamin formulations.

62

2.3 Spectrophotometric determination of vitamin C in

pharmaceuticals using Iron(III)-6-Chloro-3-hydroxy-2(4-

methoxyphenyl)-4H-chromen-4-one complex

A number of methods based on instruments67,80,128-130,142

, titrations30-32

or color

formation reactions254-256

are available in the literature for the determination of vitamin C.

Instrument- based methods are reported but a specialized and expensive equipment is

needed for these procedures. Titrimetric methods are suggested for ascorbic acid assay,

but such methods in general, lose their applicability to colored solutions due to the

interference from other reducing substances.

The purpose of the present work was to develop a simple and rapid method for the

determination of ascorbic acid. The proportionate decrease observed in the color intensity

of iron(III)-6-Chloro-3-hydroxy-2(4-methoxyphenyl)-4H-chromen-4-one (CHMC)

complex by the addition of ascorbic acid and its extraction into dichloromethane forms

the basis of the proposed method.

2.3.1 Experimental

Instrument

A Systronics spectrophotometer (model 166) with a pair of matched 1cm

quartz cells was used for measurement of absorbance.


All reagents were of analytical grade, and double distilled water was used for

preparing solutions.

63

Iron (III) solution

A (1mg ml-1




) was obtained by

appropriate dilution of the stock solution.

6-Chloro-3-hydroxy-2(4-methoxyphenyl)-4H-chromen-4-one (CHMC) solution

A 0.05% (w/v) solution of reagent was prepared by dissolving the reagent in

ethanol.

O

O

OH

OCH3

Cl

6-Chloro-3-hydroxy-2(4-methoxyphenyl)-4H-chromen-4-one (CHMC)

Ascorbic acid

Ascorbic acid solution 20 μg ml-1

was obtained by dilution of the freshly

prepared concentrated solution.

Procedure

Into a 100 ml separatory funnel, 50 μg of iron (III) solution and an aliquot of

ascorbic acid were added. After mixing the contents, 1 ml of CHMC solution was

added. The volume was made to 10 ml with water. The colored complex was extracted

64

for 1 min into 10 ml of dichloromethane. The extract was then transfered to a 10 ml

volumetric flask and its volume was made up with dichloromethane. The absorbance

of the brown complex was measured at 405nm against the reagent blank prepared

similarly. The amount of the ascorbic acid was calculated from the standard

calibration curve prepared by taking different amounts of ascorbic acid up to 8.0 μg

ml-1

and using the conditions of the procedure.

Determination of Ascorbic acid in pharmaceuticals

A known number of vitamin C tablets or capsules (5-7 items) were ground to

the powder form. An accurately weighed amount equivalent to 100 mg of ascorbic

acid was dissolved in deionized water. The solution was filtered and the filterate was

transferred to 100 ml volumetric flask. The volume was made up to the mark with

water. A lower concentration (100 μg ml-1

) was obtained by suitable dilution of this

solution. The diluted solution was analysed by the recommended procedure.


Spectral characterstics

It was observed that Iron (III) forms an extractable colored complex with 6-

chloro-3-hydroxy-2(4-methoxyphenyl)-4H-chromen-4-one. The color intensity of Fe(III)-

CHMC complex decreases with the increase in the amount of ascorbic acid which forms

the basis of proposed method. The electronic spectrum of Fe(III)-CHMC in

dichloromethane was studied along with that of reagent blank over the range 370-600 nm.

The spectrum of the complex reveals the absorption band at 403-407 nm (Fig. 09), where

the absorption due to reagent blank is negligibly small. Hence, all absorbance were

carried out at 405 nm.

65

Fig 09 Absorption spectrum of Iron(III)-CHMC complex in Dichloromethane

(Conditions: Fe(III) = 50 μg; CHMC solution = 1 ml )

A – Reagent blank against Dichloromethane


380 400 420 440 460 480 500 520 540 560 580 600 620

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

A

B

Absorb

ance

Wavelength (nm)

66

Choice of extractant

The extraction of complex was studied in to different solvents including

dichlorometane, chloroform, carbontetrachloride, benzene, xylene, iso-Amylalcohol and

n-hexane as studied (Table 13). The colored complex gets extracted in to dichlorometane,

chloroform, carbon tetrachloride and benzene. However, iso-Amyl acetate and n-hexane

were found to extract the complex incompletely. Among these solvents, dichloromethane

was found to have maximum absorbance value, so chosen for the further studies.

Table 13


Solvent Absorbance*


Chloroform 0.663


Benzene 0.443

Iso-Amyl alcohol 0.174

n-Hexane 0.078



The parameters which influence the absorbance of the complex were studied as

shown in the Table 14. In the study of each parameter, 10 ml of the aqueous phase

containing 50 μg of Iron(III) was equilibrated with an equal volume of

dichloromethane. The other conditions used therein are indicated in Table 14.

67

Effect of the reagent (CHMC) concentration

An increase in the CHMC concentration up to 0.8 ml of the reagent solution

enhances the absorbance of the colored complex which thereafter remains constant up

to 2.0 ml but there is a gradual decrease in absorbance above this concentration (Table

14, Fig.10 Curve A). Hence 1.0 ml of the CHMC solution was used for further studies.

Effect of pH

The extraction of the Fe(III)-CHMC complex was studied over a pH range 1.7-

8.4. It was observed that the complex gives maximum absorbance within pH range of

3.4-6.8 (Table 14, Fig.10 Curve B). However a decrease in absorbance is observed on

both sides of the range.


An increase in the contact time between two phases up to 0.75 min enhances

the extraction as shown by corresponding increase in the absorbance of the complex. It

remains constant up to 1.50 min of equilibration time (Table 14, Fig.11). Therefore,

equilibration time of 1 min was chosen for effecting the extraction of the complex.

68

Fig. 10 A – Effect of CHMC Concentration

B – Effect of pH

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.2

0.3

0.4

0.5

0.6

0.7

1 2 3 4 5 6 7 8 9

0.1

0.2

0.3

0.4

0.5

0.6

0.7

CHMC Concentration (ml)

Ab

so

rba

nce

BA

pH

Ab

so

rba

nce

B

A

69

Fig. 11 Effect of Equilibration Time

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

Absorb

ance

Equilibration Time (min)

70

Table 14


CHMC Conc.

(in ml)

0.2 0.4 0.6 0.8-2.0 2.5

Absorbance 0.226 0.339 0.613 0.679 0.664

Equilibration

time (in min)

0.25 0.5 0.75-1.50 1.75

Absorbance 0.324 0.547 0.679 0.662

pH 1.7 2.3 3.1 3.4-6.8 7.5 8.4

Absorbance 0.174 0.284 0.547 0.679 0.473 0.327

Conditions: Iron(III) = 50 μg ; volume of reagent (CHMC) solution = 1.0 ml ;

equilibration time = 1 min ; volume of aqueous phase = 10 ml ; volume of

dichloromethane = 10 ml; λmax = 405 nm

Calibration curve, molar absorptivity and Sandell’s sensitivity

Under the optimum conditions, Beer‟s law obedience and its range was

checked at 405 nm by adding different amount of ascorbic acid to iron(III) solution.

Beer‟s law holds good over the range 0.0- 8.0 μg ml-1

(Table 15, Fig. 12) with molar

absorptivity and Sandell‟s sensitivity of 9.581 x 103 l mol

-1 cm

-1 and 1.838 x 10

-4 μg

cm-2

respectively.

71

Fig 12 Beer‟s law curve for varied amount of ascorbic acid

0 20 40 60 80 100

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Ab

so

rban

ce

Conc. of Ascorbic Acid ( g/ 10 ml)

72

Table 15

Variation in the Absorbance with Amount of Ascorbic Acid


0 0.679

10 0.595

20 0.526

30 0.446

40 0.359

50 0.272

60 0.177

70 0.105

80 0.032

90 0.018

100 0.014


Effects of the substances commonly found in pharmaceutical formulations

were studied and the results are shown in the Table 16. These include some of the

additives, vitamins and organic acids. The data pertaining to the tolerance of each of these

substances for the determination of 50 μg ascorbic acid are as follows (mg amounts given

in the parenthesis): glycerol (500); formaldehyde (400); fructose (250); glucose, sucrose

(200); maltose (150); lactose , thiourea (100); urea (80); Cl-, SO4

2- (25); Na(I) and K(I)

(20); Ca (II), Mg (II) (15); Al(III) (10); Zn(II), benzoic acid (5); salicylic acid (2.5);

Asparatic acid, methionine, succinic acid (2.0); thiamine hydrochloride (1.5); pyridoxine

hydrochloride, glutamic acid and nicotinic acid (1.0); riboflavin (0.8); Nicotinamide (0.6);

tartaric acid (0.5); cyanocobalamin, cysteine (0.5); folic acid, citric acid (0.03).

73

Table 16



Sugars Fructose 250

Glucose, Sucrose 200

Maltose 150

Lactose 100


Asparatic acid 2.0


Pyridoxine hydrochloride, 1.0

Glutamic acid, Nicotinic acid

Methionine 2.0

Riboflavin 0.8

Nicotinamide 0.6

Cyanocobalamin, Cysteine 0.5

Folic acid 0.03

Organic acids

Benzoic acid 5.0

Salicylic acid 2.5

Succinic acid 2.0

Tartaric acid 0.5

Citric acid 0.03

Inorganic ions

Ca(II), Mg(II) 15

Al(III) 10

Zn(II) 5

Cl-, SO4

2- 25

Na(I), K(I) 20

Miscellaneous

Glycerol 500

Formaldehyde 400

Thiourea 100

Urea 80


74

Applications

The method can be applied to the analysis of commercial pharmaceutical

preparations. Vitamin C tablets and multivitamin capsules were analysed for ascorbic acid

amount by the recommended procedure. The results are mostly in close agreement with

the amount given by manufacturer‟s specification (Table 17); except for the multivitamin

formulations where a slight lower amount of the ascorbic acid was found.

Table 17



Claimed Found

1 Celin 500 498.7

2 Celin( Chewable) 200 198.6

3 Limcee 100 99.2

4 Supradyn (multivit.) 150 147.8

5 Sym-o-vit (multivit.) 75 73.3

75

2.3.3 Abstract

A simple procedure is described for the

determination of L-ascorbic acid, based on the reduction

of Iron (III) by L-ascorbic acid. The Iron(III) is

complexed with 6-chloro-3-hydroxy-2(4-methoxyphenyl)-

4H-chromen-4-one (CHMC) in acidic medium. The

absorbance is measured at 405 nm after extracting the

brown colored complex in to dichloromethane. Beer’s

law is obeyed over the concentration range up to 8.0 μg

of ascorbic acid. The method has been applied to the

analysis of various pharmaceutical formulations.

76

2.4 Spectrophotometric determination of vitamin C using Fe(II)-5-

Chloro-7-iodo-8-hydroxyquinoline complex

Many methods have been reported for the determination of ascorbic acid in

pharmaceutical preparations and food products. These include indirect

spectrophotometric methods based on the reduction of compounds such as DCIP175-180

,

Iron(III)229-248

, iodate34

, the ketone derivatisation method with o-phenylenediamine53-

57. Electrochemical, fluorimetric

53-59, kinetic

82-87, enzymatic

147-148, and

chemiluminescence69-77

methods have also been proposed. These methods have been

used to increase the analytical sensitivity for ascorbic acid and some of them have

been automated, but specialised equipments are required for these procedures.

5-Chloro-7-iodo-8-hydroxyquinoline (CIHQ) has been found to form a colored

complex with Iron (II) and Iron(III) in slightly acidic medium. However, the reaction

of Fe(III) with the 5-Chloro-7-iodo-8-hydroxyquinoline can be effectively masked by

the addition of citrate. The Fe(II)- CIHQ complex is extracted into chloroform to give

a reddish brown extract. The method based on the extraction of Iron(II)–CIHQ

complex has been worked out to look for better characteristics of a spectrophotometric

method. The detailed studies pertaining to the proposed method are presented here.

2.4.1 Experimental

Instrument

A Systronic spectrophotometer (model 166) with a pair of matched 1cm

quartz cells was used for absorbance measurements.


All reagents were of analytical grade and double distilled water was used for


77

Iron (III) solution

A stock solution of iron(III) (1 mg ml-1

) was prepared by dissolving accurately

weighed amount (0.8632 g) of Ammonium ferric sulphate in 100 ml of deionised

water containing 0.5 ml of concentrated sulphuric acid. A lower concentration (100 μg

ml-1

) was obtained by suitable dilution of the stock solution with distilled water.

5-Chloro-7-iodo-8-hydroxyquinoline (CIHQ) solution


N

OH

I

Cl

5-Chloro-7-iodo-8-hydroxyquinoline

Ascorbic acid Solution


) was used.

Potassium Citrate solution

A 5% (w/v) aqueous solution of Potassium Citrate was prepared in distilled

water.

Procedure

Into a 100 ml separating funnel, 1ml (100 μg) of Iron(III) solution was taken

and an aliquot of ascorbic acid was added. After swirling the contents, 2 ml of

78

potassium citrate was added followed by addition of 1.5 ml of the 0.05% CIHQ

solution. The volume was made to 10 ml with distilled water. The brown colored

complex was extracted in to 10 ml chloroform for 45 sec. The extract was then

transferred to 10 ml volumetric flask and its volume was made up with chloroform.

The absorbance of colored complex was measured at 485 nm against the reagent blank

prepared similarly. The content of ascorbic acid were computed from the standard

calibration curve prepared by taking different amounts of ascorbic acid upto 8.0 μg/ 10

ml and using the conditions of the procedure.

Analysis of tablets/ capsules

A known number (5-10) of vitamin C tablets/ capsules were used to get their

powder form. An accurately weighed amount equivalent to 100 mg of Ascorbic acid

was dissolved in water. The solution was filtered and the filtrate was transferred to 100

ml volumetric flask. The volume was made up to the mark with water. The working

solution (10 μg ml-1

) was prepared by dilution. A known volume of the prepared

solution was analysed for ascorbic acid contents by the recommended procedure.

2.4.2 Result and discussion

Spectral Studies

Iron(III) gets reduced easily with Ascorbic acid to iron(II) which forms an

extractable colored complex with CIHQ reagent. The electronic spectrum of Fe(II)-

CIHQ in chloroform was studied along with that of reagent blank over the range 370-

760 nm (Fig 13), which shows two absorption bands in the region of 482-488 nm and

595-600 nm. The complex absorbs strongly at 485 nm where the absorption due to

reagent blank is small. Hence, all absorbance measurements were carried out at 485

nm.

79

Fig. 13 Absorption spectrum of Iron(II)-CIHQ complex

(Conditions: Iron(III) = 100 μg; CIHQ solution = 1.5 ml )

A – Reagent blank against chloroform


400 500 600 700 800

0.00

0.05

0.10

0.15

0.20

0.25

0.30 B

A

Ab

so

rban

ce

Wavelength (nm)

80

Choice of extractant

The extractability of the complex in various solvents was studied as shown in

Table 18. The complex gets extracted into chloroform, dichloromethane, carbon

tetrachloride, benzene to give a brown colored extract in each case (extraction

decreases in that order) While it was little extracted into Ethyl acetate and n-hexane.

As the absorbance in chloroform is maximum, hence, it was chosen as an extractant.

Table 18


Solvent Absorbance*

Chloroform 0.299



Benzene 0.156

Ethyl acetate 0.098

n-Hexane 0.010


Effect of reaction variables

The various parameters which can influence the extraction of the complex

were studied. These include the changes in concentration of the reagent, effect of pH,

effect of equilibration time and potassium citrate which were studied as given here

under.

Effect of reagent concentration

The increase in the reagent (CIHQ) concentration through 1.5 ml of CIHQ

solution leads to increase in the absorbance which remains constant up to 2.0 ml of

CIHQ solution (Table 19, Fig. 14, Curve A). However, further increase in its

81

Fig. 14 A- Effect of CIHQ concentration

B- Effect of pH

0.0 0.5 1.0 1.5 2.0 2.5

0.00

0.05

0.10

0.15

0.20

0.25

0.30

2 3 4 5 6 7 8

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32 BA

B

A

pH

Absorb

ance

Absorb

ance

CIHQ Conc. (in ml)

82

Fig.15 A- Effect of equilibration time

B- Effect of Potassium citrate concentration

0 20 40 60 80 100

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

Potassium citrate Conc. (in ml)

Ab

so

rban

ce

Ab

so

rban

ce

Equilibration Time (in seconds)

BA

A

B

83

concentration causes a gradual decrease in the absorbance of the complex. Hence, 1.5

ml of the CIHQ solution was used for further studies.

Effect of pH

The extraction of the Fe(II)-CIHQ complex was studied over a pH range 2.4-

7.8. It was observed that the complex gives maximum absorbance within range of 5.5-

6.5 (Table 19, Fig. 14, Curve B). And outside this range, absorbance was found to

deviate from the optimal value.

Effect of equilibration time

Equlibration time between the two phases influences the extraction of the

complex. On increasing the contact time between two phases up to 45 sec enhances

the extraction as can be seen by corresponding increase in the absorbance of the

complex. It remains constant up to 1.0 min. of equilibration time (Table 19, Fig.15,

Curve A). Therefore, equilibration time of 45 sec was chosen.

Table 19

Optimization of Reaction Variables

CIHQ Conc.

(in ml)

0.2 0.5 1.0 1.5-2.0 2.2 2.5

Absorbance 0.034 0.131 0.255 0.299 0.295 0.292

Equilibration

time (in sec)

5 10 30 35 40-60

Absorbance 0.171 0.206 0.274 0.295 0.299

Potassium

citrate (in ml)

0.2 0.5 1.0 1.6-2.4 3.0 3.5

Absorbance 0.173 0.216 0.268 0.299 0.281 0.272

pH 2.4 3.7 4.9 5.5-6.5 6.8 7.8

Absorbance 0.139 0.210 0.273 0.299 0.279 0.246

Conditions: Iron(III) = 100 μg ; volume of aqueous phase = 10 ml ; volume of

chloroform = 10 ml; λ max = 485 nm.

84

Effect of potassium citrate solution

It is essential to add citrate solution since it helps not only in preventing the

extraction of corresponding Fe(III)-CIHQ complex but also in adjusting the optimum

pH to 5.5-6.5 (Table 19, Fig. 15, Curve B).

Beer’s law

Varied amounts of ascorbic acid were added to check the range of Beer‟s law

obedience and it was found that a linear relationship between absorbance and the

concentration of ascorbic acid holds good upto 8.0 μg/ 10ml (Table 20, Fig. 16). The

molar absorptivity and sandell‟s sensitivity at 485 nm are found to be 8.5 x 105 l mol

-

1cm

-1 and 0.2072 x 10

-3 μg cm

-2.

Table 20



0 0.003

1 0.057

2 0.123

4 0.241

6 0.356

8 0.484

9 0.512

10 0.537

12 0.559

85


0 2 4 6 8 10 12

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

so

rban

ce

Conc. of Ascorbic Acid ( g/ 10 ml)

86


The tolerance limits of diverse substances commonly encountered in vitamin C

formulations were investigated. The results are listed in the Table 21. All of the tested

vitamins and amino acids, except cysteine, are tolerated, but to varying degrees (Table

4). No interference was observed from the sugars tested. Some of the organic

componds, such as formaldehyde, glycerol, urea, benzoic acid, succinic acid, maleic

acid, citric acid and tartaric acid, were also found to be tolerated to a good amount in

the analysis for ascorbic acid content.

Table 21

Effect of Diverse Substances

Substance added Tolerance Limit (mg/ 10ml)

Sugars

Sucrose 150

Fructose, Lactose, Glucose 100

Xylose 25

Starch 20


Aspartic Acid, Glutamic Acid 1.5

Thiamine

Methionine, Nicotinamide 1.0

Pyridoxine hydrochloride

Nicotinic Acid 0.5

Cyanocobalamin 0.4

Folic Acid 0.3

Riboflavin 0.1

Cysteine 0.05

Contd.

87

Organic Acids

Succinic Acid 15

Maleic Acid, Benzoic Acid 10

Citric acid 2.0

Tartaric Acid 1.5

Cations & Anions

Mg(II) 5.0

Ca(II) 3.0

Cl- 150

SO42-

100

Miscellaneous

Formaldehyde 400

Glycerol 200

Urea 75


Applications

The proposed procedure was applied to the determination of the ascorbic acid

contents in various pharmaceutical preparations such as Limcee tablets in pure and

dosage forms, C-mac, Vetcee injection in pure form; multivitamin products such as

Innergy and toniken plus (Table 22). All these products were collected from the

market. The amount of vitamin C obtained in each case was almost the same as

claimed by the manufacturer.

88

Table 22


Sr. No. Preparation Ascorbic Acid Content per tablet/cap./inj. (in mg)

Claimed Found

1 C-mac (inj.) 40 38.5

2 Vetcee (inj.) 50 47.8

3 Limcee 500 496.9

4 Innergy 50 48.3

5 Toniken Plus 1.0 0.9

89

2.4.3 Abstract

An extractive spectrophotometric

procedure based on the complexation of reduced

Iron(II) with 5-Chloro-7-iodo-8-hydroxyquinoline

(CIHQ) for the estimation of micro amounts of

vitamin C is described. The resulting complex is

extracted into chloroform to give a reddish brown

extract which shows an absorption band at 485 nm.

Linear relationship between absorbance and

concentration of ascorbic acid is observed up to 0.8 μg

ml-1. Interference studies of different substances

including sugars, vitamins and amino acids, metal

ions and organic acids were carried out. The utility of

the method was tested by analysing some of the

marketed products of vitamin C.

90

2.5 Use of calmagite in the determination of vitamin C involving

Ce(IV)-Ce(III) redox couple

Many of the colorimetric methods developed for the determination of ascorbic

acid involve iron(III)-iron(II)238,248,253

redox couple. Though some of them are quite

sensitive yet some of these methods cannot be used for routine analysis due to their

associated problems. Attempts have been made to improve the desirable

characteristics of such spectrophotometric methods using other redox couples such as

V(V)-V(VI)217,218

, Cu(II)-Cu(I)193,194,200

and Cr(VI)-Cr(III)192,174

but without much

success. We also thought of using a different redox couple in devising a method for

the estimation of ascorbic acid. During our preliminary investigation, we found the

suitability of the Ce(IV)-Ce(III) redox couple for the determination of ascorbic acid.

The enediol [-CH(OH)=C(OH)-] present in ascorbic acid is responsible for its

reducing properties. The reduced cerium(III) gives the formation of an brown colored

complex with calmagite in the presence of pyridine that has been used for the

development of proposed method. The detailed studies pertaining to the optimization

of the conditions affecting the absorbance of the complex, construction of calibration

curve, effect of foreign substances and its applicability to pharmaceutical products are

presented here.

2.5.1 Experimental

Instrument

A pH meter (model HPG-2001 A) and systronics spectrophotometer (model-

166) with a pair of matched 1cm quartz cells were used for pH and absorbance studies.



preparing solution.

91


A fresh stock solution of ascorbic acid was prepared by dissolving 0.100 g of

ascorbic acid in 100 ml of deionized water to get ascorbic acid solution of

concentration (1mg ml-1

). A lower concentration (10 μg ml-1

) was obtained by suitable


Cerium(IV) solution

A (1mg ml-1

) solution of cerium(IV) was prepared by dissolving 0.4514 g of

Ceric ammonium sulphate in 100 ml of deionized water containing 1 ml of

concentrated sulphuric acid. A lower concentration (200 μg ml-1

) was obtained by


Calmagite (3-Hydroxy-4-[(2-hydroxy-5-methylphenyl)azo]-1-naphthalene

sulfonic acid) solution

A 0.02% (w/v) solution was obtained by dissolving the reagent in deionized

water.

S

O

O

HO N

N

CH3

HO

OH

Calmagite

Pyridine solution

A 1 % (v/v) solution of pyridine was prepared in distilled ethanol.

92

Procedure

To the 200 μg of cerium (IV) solution taken in a separatory funnel, an aliquot

of ascorbic acid was added for the reduction of Cerium(IV) to Cerium(III). The

contents were swirled and 1 ml of reagent (calmagite) solution was added followed by

addition of 0.5 ml of pyridine solution. The aqueous volume was made to 10 ml with

water and the brown colored complex was extracted with 10 ml of dichloromethane

for 0.5 min. The two layers were allowed to separate and the extract was taken in a 10

ml measuring flask. The volume was made up to mark with dichloromethane. The

absorbance of the brown complex was measured at 485 nm against a reagent blank.

The amount of ascorbic acid was calculated from the calibration curve prepared by

taking different amounts of ascorbic acid up to 6 μg ml-1

.

Analysis of pharmaceutical products (tablets /capsules)

Analysis of tablets

A known weight of the powder obtained by crushing (5 or 10) tablets

equivalent to 1 mg ml-1

of ascorbic acid, was dissolved in water. If necessary, the

solution is filtered through a whatmann filter paper no. - 41. The working solution was

made by suitable dilution to get 100 μg ml-1

of ascorbic acid. It was then analysed for

ascorbic acid content by the proposed procedure.

Analysis of capsules

An accurately weighed amount of the capsule content equivalent to 1 mg ml-1

of ascorbic acid was dissolved in a 100 ml measuring flask. The volume was made up

to mark with deionized water. A 100 μg ml-1

solution was obtained by suitable dilution

of the stock solution. The diluted solution was analysed with recommended method.

93


Spectral studies

Cerium(IV) gets reduced easily with ascorbic acid to cerium(III) which forms

an extractable brown colored complex with calmagite reagent in the presence of

pyridine . The electronic spectrum of cerium(III)-calmagite complex was studied over

the range 370-600 nm (Fig. 17), which shows the absorption band at 483-487

nm,where absorption due to reagent blank is negligible. Hence, all absorbance

measurements were carried out at 485nm.

Choice of solvent

The extraction behaviour of the complex was studied into different solvents as

shown in Table 23. The solvents tested include dichloromethane, chloroform, carbon

tetrachloride, benzene, xylene, toluene, cyclohexane and iso-amylalcohol. Among the

solvents tested, dichloromethane and chloroform were found to extract the complex,

while it was little extracted into other solvents. Dichloromethane was chosen as an

extractant because of the higher absorbance.

Table 23


Solvent Absorbance*


Chloroform 0.113


Iso- Amylalcohol 0.037

Xylene 0.012

Benzene 0.010

Toluene 0.010

Cyclohexane 0.008


94

Fig.17 Absorption spectrum of Cerium(III)-Calmagite complex

(Conditions: Ce(IV) (200 μg) = 1 ml; Calmagite solution (0.02 % w/v) = 1.0 ml ;

pyridine solution (1 % v/v) = 0.5 ml; Vol. of aqueous = Vol. of DCM = 10 ml)

A– Complex against reagent blank

B – Reagent blank against dichloromethane

400 450 500 550 600

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Ab

so

rba

nce

Wavelength (nm)

95

Effect of reaction variables

The parameters which affect the extraction and the absorbance of the complex

were studied as shown in Table 24. In the study of each parameter, 10 ml of aqueous

phase containing 200 μg of cerium(IV) and 40 μg of ascorbic acid and 0.5 ml of

pyridine was equilibrated with equal volume of dichloromethane. The other conditions

used therein are indicated at the bottom of the Table 24.

Effect of reagent (calmagite) concentration

An increase in the amount of calmagite concentration up to 1 ml enhances the

absorbance of the complex, which remains the same up to 2 ml of its solution (Table

24, Fig. 18, Curve A). But further increase in its concentration caused gradual

decrease in absorbance. Hence, 1 ml of the reagent solution that gives highest

absorbance was used for subsequent studies.

Table 24


Calmagite conc.

(in ml)

0.5 1.0-2.0 2.5 3.0

Absorbance 0.097 0.158 0.147 0.140

pH 2.2 3.4 3.9-5.8 6.6

Absorbance 0.110 0.140 0.158 0.136

Equilibration time

(in min)

0.25 0.5-1.5

Absorbance 0.126 0.158

Pyridine conc.

(in ml)

0.2 0.4-0.8 1.0 1.2

Absorbance 0.113 0.158 0.152 0.147

Conditions: Cerium(IV) = 200 μg ; volume of ascorbic acid (20 μg ml-1

) solution =

2.0 ml ; volume of aqueous phase = 10ml ; volume of dichloromethane =10 ml; λmax

= 485 nm.

96

Fig. 18 A- Effect of calmagite concentration

B- Effect of pH

0.5 1.0 1.5 2.0 2.5 3.0

0.09

0.10

0.11

0.12

0.13

0.14

0.15

0.16

2 3 4 5 6 7

0.11

0.12

0.13

0.14

0.15

0.16

Concentration of reagent (ml)

Ab

so

rban

ce

A

B

BA

Ab

so

rban

ce

pH

97

Fig.19 A- Effect of equilibration time

B- Effect of pyridine concentration

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0.12

0.13

0.14

0.15

0.16

0.17

0.2 0.4 0.6 0.8 1.0 1.2

0.11

0.12

0.13

0.14

0.15

0.16

B

B

A

A

Absorb

ance

Equilibration time (min)

pyridine concentration (ml)

Absorb

ance

98

Effect of pH

The pH of the aqueous solution was varied over the range 2.2-6.6 (Table 24,

Fig. 18, Curve B) which was adjusted with 0.1N hydrochloric acid and 0.1N sodium

hydroxide. The optimum pH range giving higher absorbance is 3.9-5.8.

Effect of pyridine

The addition of pyridine is essential as the complex formation occurs only in

the presence of pyridine. In case there is no addition of pyridine, the absorption band

at 485 nm is not observed. The absorbance of the complex increases with the increase

in pyridine concentration up to 0.4 ml, thereafter it remains constant up to 0.8 ml

(Table 24, Fig 19, Curve B). Further addition of pyridine is responsible for a gradual

decrease in respective absorbance. Hence, further studies were made with the addition

of 0.5 ml of pyridine solution.

Effect of equilibration time

The aqueous phase containing the complex was equilibrated with 10 ml of

dichloromethane for different intervals of time (Table 24, Fig. 19, Curve A) and 0.5

min was found sufficient for the quantitative extraction of complex into non-aqueous

phase and which remains constant upto 1.5 min of equilibration time.

Beer’s law and statistical data

Under the optimum conditions, Beer‟s law plot was constructed at 485 nm by

adding different amount of ascorbic acid to Cerium(IV) solution and a linear

relationship between absorbance and concentration of the analyte was observed over

the range 0.5-6.0 μg ml-1

(Table 25, Fig. 20) of ascorbic acid. The calculated molar

absorptivity and sandell‟s sensitivity are 6.957 x 103 l mol

-1 cm

-1 and 0.02532 μg cm

-2

respectively.

99


0 10 20 30 40 50 60 70 80 90

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Absorb

ance

Concentration of Ascorbic acid ( g/10ml)

100

Table 25

Effect of Ascorbic Acid Concentration on the Absorbance of the Complex


10 0.004

20 0.057

30 0.107

40 0.158

50 0.219

60 0.283

70 0.290

80 0.263

Effect of foreign substances

The possible interference of some ingredients likely to be formulated along

with vitamin C in pharmaceutical products was investigated. Such interference studies

were carried out in the determination of 40 μg of ascorbic acid. No interference was

observed from sugars at the levels indicated in the Table 26. However, among

vitamins and amino acids, only folic acid, cysteine and asparatic acid were found to

interefere. Except citric acid, organic acids tested do not interfere. Other substances as

mentioned under the heading „Miscellaneous‟ are also tolerated.

The tolerance limits of different substances are as: 250 mg of formaldehyde;

150 mg each of glycerol and starch; 100 mg of starch and maltose; 200 mg of glucose

and fructose, 50 mg each of lactose and xylose; 80 mg of urea; 60 mg of mannose; 30

mg of maleic acid; 20 mg of benzoic acid; 8 mg each of succinic acid and tartaric acid;

5 mg of salicylic acid; 1.4 mg each of methionine and riboflavin; 1 mg each of

thiamine hydrochloride and Nicotinamide; 0.5 mg each of citric acid and F- ; 0.8 mg of

pyridoxine hydrochloride; did not interfere with the determination.

101

Table 26



Sugars


Sucrose 150

Maltose 100

Mannose 60

Lactose, Xylose 50


Nicotinic acid 2.0

Methionine, Riboflavin 1.4

Cyanocobalamin 1.2

Thiamine hydrochloride, Nicotinamide 1.0


Glutamic acid 0.5

Cysteine 0.4

Asparatic acid 0.2

Folic acid 0.06

Organic acids

Maleic acid 30

Benzoic acid 20

Succinic acid,Tartaric acid 8

Salicylic acid 5

Citric acid 0.5

Cations and anions

Na(I), K(I) 40

Zn(II), Al(III) 0.2

Cu(II) 0.1

Co(II), Mn (II) 0.05

Ca(II),Mg(II) 0.03

Cl-, SO4

2- 60

F-

0.5

Miscellaneous

Formaldehyde 250

Glycerol 150

Starch 100

Urea 80


102

Applications

The proposed method for the determination of vitamin C is sufficiently

sensitive and selective. The utility of the method was assessed by the analysis of the

various vitamin C tablets as well of multivitamin products (Table 27) containing

vitamin C as ingredient. The values found are in close agreement with the nominal

values.

Table 27



Claimed Found

1 Celin 500 489.6

2 Chewcee 500 488.7

3 Cell C 100 99.4

4 C-Vit. 150 147.8

5 Polybion 150 146.9

6 Celin -chewable 200 198.8

103

2.5.3 Abstract

An extractive spectrophotometric method for

the determination of micro amounts of ascorbic acid

has been developed by exploiting its reducing nature.

Ce(IV) is converted to Ce(III) by the addition of

ascorbic acid, which forms a brown colored complex

with calmagite and pyridine. The absorbance is

measured at 485 nm after extracting the colored

complex into dichloromethane. Beer’s law holds good

in the concentration range of 0.5-6 μg ml-1. The effect

of various substances such as vitamins, amino acids,

sugars and other additives has been investigated. The

applicability of the method was tested by analysing

pure vitamin C and multivitamin pharmaceuticals.

104

2.6 Spectrophotometric determination of vitamin C using Iron(II)-

Bathophenanthroline complex

A number of spectrophotometric methods have been developed using a wide

variety of reagents but are associated with shortcomings of one type or the other, for

example methods using 2,2‟-bipyridyl229-231

, 1,10-phenanthroline238-240

, ammonium

molybdate86,206-208

, folin-coicalteu216

etc. are time consuming; these methods need at

least 1 h for a single determination. Methods based on ferrozine254-256

,

dimethoxydiquinone184

and 2,3,5-triphenyltetrazolium chloride219

must indure many

interferences.

While seeking for the development of a method with improved characteristics,

bathophenanthroline has been found to be a suitable reagent that forms a red colored

complex with reduced iron(II) over the pH range 4.0-5.5. The method based on the

extraction of iron(II)-bathophenanthroline complex in dichloromethane provides the

desirable features of simplicity and rapidity besides having better sensitivity and

selectivity.

2.6.1 Experimental

Instrument

A systronics spectrophotometer (model-166) with a pair of matched 1cm

quartz cells was used for absorbance measurements.




Buffer solution

Acetate buffer solution (pH 5.0) was prepared by mixing 35.7 ml of 1M acetic

acid and 64.3 ml of 1M sodium acetate (Trihydrate) solution.

105



) was used. A lower



Iron (III) solution

A (1 mg ml-1

) iron (III) solution was prepared by dissolving accurately

weighed amount of ammonium ferric sulphate in 100 ml of deionised water containing

0.5 ml of concentrated sulphuric acid. A lower concentration (100 μg ml-1

) was

obtained by dilution of the stock solution.

Bathophenanthroline (Bphen) solution


N N

Bathophenanthroline

Procedure

An aliquot of ascorbic acid was added to the 100 µg of iron(III) solution taken

in a separatory funnel followed by addition of 2 ml of acetate buffer solution for

adjusting the pH in the range of 4.0-5.5 and 1ml of bathophenanthroline solution.

Enough water was added to make the aqueous phase to 10 ml. The resulting red

colored complex was extracted for 30 sec with 10 ml of dichloromethane (DCM). The

coloured extract was taken into a 10 ml volumetric

106

flask and the volume was made up to the mark with DCM, if required. The absorbance

of the red colored complex was measured at 485 nm against the reagent blank

prepared similarly and the vitamin C contents is determined from the standard

calibration curve prepared by taking different amounts of ascorbic acid up to 12 μg/

10ml and using the optimum conditions of the procedure.

Determination of ascorbic acid in pharmaceutical products (tablets /capsules)

The tablets or capsules (5-10 items) were crushed to the powder form. An

accurately weighed amount equivalent to 100 mg of ascorbic acid was dissolved in

deionized water. The solution was filtered into a 100 ml volumetric flask and made up

to mark with deionized water. The working solution of lower concentration (10 μg ml-

1) was obtained by suitable dilution of this solution. The diluted solution was analysed

by the proposed procedure.


Spectral studies

Iron(II) forms an extractable red colored complex with bathophenanthroline in

acidic medium. Preliminary investigations revealed a quantitative increase in color

intensity of iron(II)-Bathophenanthroline complex by the addition of increasing

amount of ascorbic acid. The absorption spectrum of iron(II)-Bathophenanthroline

complex was studied over the range 350-720 nm, which shows the absorption band at

480-487 nm (Fig. 21), where absorption due to reagent blank is very small. Hence, all

absorbance measurements were carried out at 485 nm.

Choice of solvent

Various solvents were tested to extract the Fe(II)-Bathophenanthroline complex as

shown in Table 28. The solvents include dichloromethane, chloroform, carbon

tetrachloride and benzene. However, n-Hexane, iso-Amyl acetate were found to

extract the complex partially. Dichloromethane was chosen as an extractant because of

the highest absorbance in this solvent.

107

Fig. 21 – Absorption spectrum of Iron(II)-Bathophenanthroline complex (Conditions:

Iron(III) =100 μg; Bathophenanthroline solution = 1.0 ml )

A – Reagent blank against dichloromethane

B – Complex against reagent blank.

350 400 450 500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

0.25

B

A

Ab

so

rban

ce

Wavelength (nm)

108

Table 28


Solvent Absorbance*


Chloroform 0.227


Benzene 0.083

n-Hexane 0.025

iso-Amyl acetate 0.017


Effects of reaction variables

The various parameters which can influence the extraction of the complex and

absorbance were studied (Table 29). These include the change in concentration of the

reagent, equilibration time and pH of the medium. In the study of each parameter, 10

ml of aqueous phase containing 100 μg of iron(III) and 10 μg of ascorbic acid was

equilibrated with equal volume of dichloromethane.

Effect of Bathophenanthroline concentration

An increase in the Bathophenanthroline concentration up to 0.8 ml of the

reagent solution enhances the absorbance of the complex which thereafter remains the

same up to 1.3 ml but there is a gradual decrease in absorbance above this

concentration (Table 29, Fig. 22 Curve A). Hence, 1.0 ml of the Bathophenanthroline

solution was used for further studies.

109

Table 29


BPhen conc.

(in ml)

0.4 0.6 0.8-1.3 1.5 2.0

Absorbance 0.127 0.217 0.244 0.242 0.239

Amount of buffer

(in ml)

0.0 0.5 1.0-2.5 3.0

Absorbance 0.203 0.241 0.244 0.237

pH 2.0 3.1 4.0-5.5 5.7 6.4

Absorbance 0.226 0.237 0.244 0.242 0.194

Equilibration time

(in sec)

5 10 20 30-60

Absorbance 0.069 0.153 0.208 0.244

Conditions: Iron(III) (100 μg ml-1

) = 1 ml , Ascorbic acid (10 μg ml-1

) = 1 ml,

Volume of aqueous phase = Volume of dichloromethane =10 ml , λmax = 485 nm

Effect of pH

The extraction of the Fe(II)- Bathophenanthroline complex was studied over a

pH range 2.0-6.4. It was observed that the complex gives maximum absorbance within

range of pH 4.0-5.5 (Table 29, Fig. 22, Curve B). However, a decrease in absorbance

is observed on going to either side of the range.


An increase in the time contact between two phases up to 30 sec enhances the

extraction as observed by corresponding increase in the absorbance of the complex. It

remains constant up to 60 sec of equilibration time (Table 29, Fig. 23). Therefore,

equilibration time of 30 sec was chosen.

110

Fig. 22 A Effect of Bathophenanthroline concentration

B Effect of pH

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.262 3 4 5 6 7

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

Ab

so

rban

ce

Ab

so

rban

ce

Conc. of Bathophenanthroline (in ml)

B

B

A

A

pH

111

Fig.23 Effect of equilibration time

0 10 20 30 40 50 60

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.260.0 0.5 1.0 1.5 2.0 2.5 3.0

0.20

0.21

0.22

0.23

0.24

0.25

Amount of Buffer solution (ml)

Ab

so

rba

nce

Ab

so

rba

nce

Equilibration time (sec)

BA

B

A

112

Calibration curve

A linear relationship between absorbance and concentration of the analyte was

found to hold good within the range 0.0-1.2 µg ml-1

under the optimum conditions of

the procedure (Table 30, Fig. 24). The calculated molar absorptivity and sandell‟s

sensitivity are found to be 4.296 × 104 l mol

-1 cm

-1 and 0.0041 µg cm

-2 respectively.

Table 30



2 0.042

4 0.097

6 0.146

8 0.192

10 0.244

12 0.294

14 0.316

16 0.320


The studies pertaining to the influence of possible constituents of vitamin C

formulations were carried out and the resulting data are summarized in Table 31.

In the determination of 10 μg ml-1

of ascorbic acid, different substances which are

tolerated to different degrees include (mg in parenthesis) sugars, amino acids,

vitamins, organic acids, inorganic cations and anions. Sugars are tolerated at the levels

indicated but the tolerance limit for the studied vitamins and amino acids is not high

especially with cysteine and riboflavin. Among the tested organic acids citric acid was

found to interfere. For the tested cations and anions, Cu(II) and Al (III) are tolerated in

traces whereas sodium sulphite and nitrate interfere seriously with the determination.

Some other substances as mentioned under the heading „miscellaneous‟ are also

tolerated.

113

Fig. 24 Beer‟s law curve for ascorbic acid.

0 2 4 6 8 10 12 14 16 18

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Absorb

ance

Conc. of Ascorbic acid ( g/ 10ml)

114

Table 31



Sugars Sucrose 200

Glucose, Fructose, lactose 100

Starch, mannose 50

Xylose 20


Asparatic acid 2.0

Methionine, Thiamine 1.5

Glutamic acid, Nicotinic acid 1.0

Nicotinamide, Pyridoxine hydrochloride 0.5

Cyanocobalamin 0.4

Folic acid 0.2

Riboflavin 0.03

Cysteine 0.02

Organic acids

Benzoic acid 10

Succinic acid 20

Maleic acid 15

Tartaric acid 1.5

Salicylic acid 2.0

Citric acid 0.1

Cations and Anions

Ca(II), Mg(II) 20

Al(III) 0.1

Cu(II) 0.3

Cl- 60

SO32-

0.02

NO3- 0.05

Miscellaneous

Formaldehyde 300

Glycerol 250

Urea, Thiourea 50


115

Applications

The proposed method for the determination of vitamin C is sufficiently

sensitive and selective. The utility of the method was assessed by the analysis of the

various pure form, multivitamin products (Table 32) containing vitamin C as an

ingredient. The values found were in close agreement to the prescribed values. The

other features of the method include simplicity and rapidity.

Table 32


Sr.No. Preparation Ascorbic Acid Content per tablet (in mg)

Claimed Found

1 Ascorbic acid (C-Mac) inj. 40 38.8

2 Ascorbic acid (Vetcee) 50 49.0

3 Limcee 500 498.7

4 Innergy-24 50 47.4

5 Tonikem plus 01 0.92

116

2.6.3 Abstract

A spectrophotometric method has been

developed for the determination of vitamin C in

a variety of samples. The procedure is based on

the complexation of reduced iron(II) with

Bathophenanthroline followed by its extraction

into dichloromethane. The absorbance is

measured at 485 nm. Beer’s law is obeyed up to

1.2 µg ml-1 of ascorbic acid. Interference effects of

various substances including sugars, vitamins,

amino acids, inorganic cations and anions and

some organic substances have been studied.

2. methods for spectrophotopmeric determination of vitamin...

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