1

1.1 General Introduction

Vitamins are organic molecules required in small quantities1 for the normal

growth and maintenance of human beings. Most of them are aromatic substances

except pantothenic acid which is a straight chain compound. Generally plants

products are rich sources of vitamins, but in animal organisms they are formed

only as a result of food intake or due to anabolic activity of the microorganisms

living in the intestinal tract. Vitamins are usually concentrated in those animal and

plant tissues which are most active metabolically. Thus, liver or kidney are more

potent sources of vitamins than muscles, skin or other parts. Most of them carry

out their functions in the form of coenzymes or prosthetic group of enzymes.

Together with certain amino acids, the vitamins constitute a total of 24 organic

compounds that have been characterized as dietary essential. Vitamins play a vital

role in the metabolic processes of body (i.e. maintenance, growth, development

and/or production). But their absence or underutilization may cause a specific

deficiency syndrome.

Foods obtained from plants and animals are most important sources of

vitamins in the daily diet of human beings. However, the vitamins are unevenly

distributed among the various food sources of plant and animals origin. It is

possible to eliminate the vitamin deficiency diseases by the intake of synthetic

vitamins. They are divided in to two categories depending on their solubility. The

water soluble vitamins are vit.B1, vit.B2, vit.B3, vit.B5, vit.B6, vit.B12, vit. C,

folic acid and vit.H (biotin) whereas fat soluble vitamins are vit. A,vit. D,

vit.E.and vit.K. The important features of different vitamins and their vitamers are

summarized2,3

in the Table 1. Among different water soluble vitamins, vitamin C is

of greater interest because of its wide spread consumption in the form of

vegetables, fresh fruits or pharmaceuticals products since it is considered essential

for the development and regeneration of muscles, bones, teeth, skin and immune-

related functions. Wide spread distribution of vitamin C is found in nature

especially in plant materials: citrus fruits like orange , lemon and tomatoes,

potatoes and fresh green vegetables are some of the excellent sources of vitamin C.

2

Table 1

Important Features of Different Vitamins

Vitamin Vitamers Dietary Sources Physiological Functions Deficiency Syndrome

Fat Soluble Vitamins

Vit. A Retinol Milk, butter, cheese, egg yolk, Visual pigments epithelial cells Xerophthalmia

Retinal fatty fish, green vegetables, differentiation.

Retinoic acid yellow to red fruits, especially carrots.

Vit. D Cholecalciferol Fatty fish, margarine and fortified Calcium homeostasis Rickets and

Ergocalciferol milk. Osteomalacia

Vit. E α-Tocopherol Vegetables oils Membrane antioxidant Multiple effects

γ- Tocopherol

Vit. K Phylloquinones Green leafy vegetables and Blood clotting, calcium Reduced coagulability,

Menaquinones liver metabolism increased bleeding

Menadione tendency

Contd.

3

Water Soluble Vitamins

Vit. B1 Thiamine Seeds and grains Coenzyme for decarboxylations Beri-beri, cardionegaly

of 2-keto acids and transketolations

Vit. B2 Riboflavin Liver, milk, cheese, yeast Coenzyme in redox reactions of Chelosis, glossitis, local

Fatty acids, the TCA cycle inflammations

Vit.B3 Pantothenic acid Yeast, egg, meat, liver, honey, Coenzyme in fatty acid metabolism Impaired adrenal

Sugarcane functions, cardiovascular

Instability

Vit.B5 Nicotinic acid Meat, fish, yeast, soyabean Coenzyme for several Pellagra

(Niacin) Nicotinamide dehydrogenases

Vit.B6 Pyridoxal Liver, meat, fruit, leafy Coenzyme in amino acid meta- Dermatitis, neurological

Pyridoxine vegetables bolism disorders

Pyridoxamine Contd.

4

Vit. B12 Cobalamin Milk, meat, liver Coenzyme in metabolism of Pernicious anaemia

propionate, amino acids

Vit. C Ascorbic acid Citrus fruits, potatoes, green Reductant in hydroxylation Scurvy

leafy vegetables reactions and in the metabolism

of drugs and steroids

Vit. H Biotin Liver, kidney, yeast extracts Coenzyme for carboxylations Dermatitis

-- Folic acid Liver, green vegetables Coenzyme in single-carbon Macrocytic anaemia

metabolism

5

high dietary contents of vitamin C are also found in Brussels sprouts, parsley, black

currants, kale, spinach, rose hips, coriander, papaya and strawberries. Animal tissues

also contain small but a definite amount of vitamin C. Increased amounts are also

found in various organs/glands such as Ham, liver, chicken, beef, adrenal gland and

thymus. Human milk also contains considerable amount of vitamin C. The average

values for vitamin C found in various food stuffs3,4

are given in the Table 2. Vitamin C

occurs in plants predominantly as such. In animals and probably in plant tissues

vitamin C exists in two forms: ascorbic acid and dehydroascorbic acid which are

present in an equilibrium with each other. The percentage present of the oxidised and

reduced forms varies considerably according to the tissues and other physiological

factors.

All animals except man, other primates and guinea pigs are able to synthesize

ascorbic acid, of their requirement from D-glucoronic acid, a derivative of glucose,

through the pathways shown in scheme –I.

The direct oxidative pathway for the glucose is utilized in animals that make

ascorbic acid. Gulonolactone oxidase is compromised or absent in animals that cannot

make ascorbic acid. In plants and bacteria that make L- ascorbic acid (pathway

through galactose, mannose), in addition to D-glucose can contribute to ascorbic acid

production.

Vitamin C is the generic descriptor for all the compounds exhibiting

qualitatively the biological activity of ascorbic acid. The chemical name for ascorbic

acid is 2,3-didehydro-L-threo-hexano-1,4-lactone, while the other terms include

cevitamic acid, antiscorbutic acid, hexuronic acid, L-xyloascorbic acid, vitamin C. It is

a stable, odourless and water soluble crystalline solid. Ascorbic acid exists in two

enantiomeric forms D- and L- but only the later form is responsible for vitamin C

activity. The acidic behaviour is due to enolic hydrogen at C-3 which readily ionizes

to produce ascorbateion. Ascorbic acid is a moderate reducing agent which is oxidised

under mild conditions to DHAA (dehydroascorbic acid) via radical intermediate

semidehydroascorbic acid (monodehydroascorbic acid).

6

Table 02

Vitamin C in Selected Food Stuffs

Sources Edible Portion (mg/ 100 g) Sources Edible Portion (mg/ 100 g)

Animal products

Ham 20–25 Pear 2–5

Liver, chicken 15–20 Plums 2–3

Beef 10

Kidney, chicken 6–8 Vegetables

Gizzard, chicken 5–7 Brussels sprout 100–120

Heart, chicken 5 Broccoli 80–90

Human milk 3–6 Kale 70–100

Lobster 3 Cauliflower 50–70

Scrimp muscle 2–4 Chive 40–50

Crab muscle 1–4 Spinach 35–40

Pork 1–2 Cabbage 30–70

Veal 1–1.5 Radish 25

Cow milk 0.5–2 Asparagus 15–30

Fruits Eggplant 15–20

Rose hips 250–800 Tomato 10–20l

Currant, black 150–200 Beans 10–15

Kiwi fruit 80–90 Onion 10–15

Strawberry 40–70 Pea 8–12

Lemon 40–50 Beet 6–8

Grapefruit 30–70 Carrot 5–10

Orange 30–50 Potato 4–30

Currant, red 20–50 Spices and Condiments

Cherry 15–30 Parsley 200–300

Pineapple 15–25 Pepper 150–200

Mango 10–15 Coriander (spice) 90

Melons 9–60 Horseradish 45

Blackberry 8–10 Chicory 33

Banana 8–16 Garlic 16

Apple 3–30 Lettuce 10–30

Leek 5

7

D-Glucose

C

C

C

C

C

HOOC

OHH

H OH

HHO

OHH

H O

NADPH + H+

NADP+

Glucoronate reductase

C

C

C

C

C

HOH2C

HOH

H OH

HHO

HOH

OHO

C

C

C

C

HOH2C

HOH

H

HHO

HOH

O

O

C

C

C

C

HOH2C

HOH

H

HHO

O

OO

Aldonolactonase

L-Gulonic acidD-Glucoronic acid

L-Gulonolactone

Gulonolactone oxidaseO2

2-Keto-L-Gulonolactone

C

C

C

C

HOH2C

HOH

H

HO

OH

O

O

L-Ascorbic acid

D- Glucose-6-P

D- Fructose-6-P

D- Mannose-6-P

D- Mannose-1-P

GDP-D-Mannose

GDP-L-Galactose

L- Galactose

L- Galactose-1,4-lactone

Scheme - I Biosynthesis pathway for the synthesis of Ascorbic acid

8

Ascorbic acid exists in several different forms. The two predominant forms

and some of their associated oxidation products are shown in the scheme - II. In

solution, ascorbic acid probably exists as the hydrated semiketal.AscH2 (reduced

ascorbic acid)↔Asc [-] (ascorbate radical) ↔DHAsc (dehydroascorbic acid). The

most important chemical property of ascorbic acid is the reversible oxidation to

semidehydro-L-ascorbic acid and oxidation further to dehydro-L-ascorbic acid, this

property is the basis for its known physiological activities. The three forms (ascorbic

acid, semidehydroascorbic acid, dehydroascorbic acid) comprise a reversible redox

system making the vitamin, an effective quencher of free radicals. The above

transformation is a reversible step but further conversion of DHAA to DKGA and

other minor products is an irreversible reaction which is enhanced by alkaline pH and

metals (Cu,Fe). Above pH 7.0, alkali-catalysed degradation results in over 50

compounds mainly mono-, di-, and tricarboxylic acids.

HO

HO

OH

OO

OH

HO

OH

OO

O-

O

-e-- 2H+

+e-+ 2H+

-e-

+e-

O

HO

OH

OO

O

AscH2 Asc[ ] DHAsc

2,3-Diketo-L-gulonic acid

CO2

L-Xylonic acid

+

L-Lyxonic acid

Oxalic acid

L-Threonic acid

CO2

L-Xylose

Chemical forms of Ascorbic acid, major metabolites and degradation products

Scheme - II

9

The vitamin can be stabilized in biological samples with trichloroacetic acid or

metaphosphoric acid. Therefore, procedure for stabilization of vitamin C in biological

specimen requires acidification or addition of some reducing agents or metal chelator.

CH2OH

C

C

C

C

OHC

HHO

H OH

HHO

HHO

D-Glucose

H2

Cu-Cr

CH2OH

C

C

C

C

HOH2C

HHO

H OH

HHO

HHO

(+) -Sorbitol

Acetobactor

Suboxydans

OH

HOH2C

HO

CH2OHH

OH H

HO

(-) - Sorbose

OH

H2C CH2OHH

H

OMe2C

O

O

CMe2

O

2Me2CO,

H2SO4

(i) KMnO4,

NaOH

(ii) H2SO4

COOH

C

C

C

C

HOH2C

HOH

H OH

HHO

O

CHCl3 Soln

HCl

C

C

C

C

HOH2C

HOH

H

HO

OH

O

O

L- Ascorbic acid 2- Ketogulonic acid Diacetone-(-)-sorbose

Commercial Synthesis of L- Ascorbic acid

Scheme - III

10

Because DHAA is readily reduced in vivo, so it possesses vitamin C activity whereas

diketogulonic acid has no such activity. Commercialy vitamin C is prepared5 from D-

glucose which is converted to L-ascorbic acid involving the reaction steps as shown in

the scheme - III.

The metabolic roles of vitamins are primarily related to their co-enzyme

derivatives, but in the case of vitamin C it does not participate as coenzyme in a strict

sense but as a reversible biological reductant. Therefore, unlike other water soluble

vitamins, dietary vitamin C does not require conversion into a coenzyme derivative in

order to function in metabolic reactions. It is considered to be the most versatile and

effective water soluble antioxidant. Vitamin C participates in collagen formation6, a

critical step in wound healing. Vitamin C rich food reduces the risk of cancers of

intestinal tract7 and it may enhances the functions of immune modulators such as

blood histamine, prostaglandin, prostacyclin and B- and T-cell cyclic nucleiotides8,9

.

Supplement of vitamin C may reduce risk factor for cardiovascular diseases10,11

by

increasing levels of high density lipoproteins and by reducing cholesterol. Adequate

vitamin C status has been associated with a reduced risk of cataract and maintenance

of healthy bones. It is also involved in the synthesis of various hormones such as

corticosteroids and aldosterone in adrenal cortex12

. Ascorbic acid is specific in the

treatment of scurvey. Also it is an effective cure or preventive of common cold and

from cell culture studies it has been shown that ascorbic acid itself and in combination

with copper ions is selectively toxic to melanoma cancer cell (anticancerous agents). A

high vitamin C intake reduces the risk of periodontal diseases. Ascorbic acid nutriture

has also been found to effect fertility13

via its role in cellular oxidant defence and

hormone production.

There has been a great difficulty is establishing human requirement for vitamin

C. However, the following dietary allowances have been recommended.

35-40 mg per day for children‟s and adolescents

45 mg per day for adults (men and women)

85 mg per day for pregnant lactating women

11

But, individuals with kidney stones or renal diseases14

are advised to avoid

high intake of vitamin C. Moreover, the excessive intake of vitamin C is harmful since

its megadoses results15

in nephrolithiasis, reproductive failure, vitamin C dependency,

inactivation of vit. B12, loss of folic acid in urine, allergies, diarrhoea, aciduria

(oxalic, folic, uric), possible damage to β-cells of pancreas, decreased insulin

production by dehydroascorbic acid and thrombosis etc. It is not possible to establish

with any certainty an upper level of intake for supplementary vitamin C. However,

expert bodies have suggested that intake of no more than 1000 mg/ day for adults

would be prudent16-17

.

1.2 Review of the methods for vitamin C determination

Vitamin C occurs in nature in a variety of samples such as fruits, vegetables

and animal products, as an essential ingredient, a stabilizer for vitamin B complex, as

a cofactor for the activity of enzymes and as an antioxidant. Keeping in view its

importance, the analysis of food products and pharmaceuticals containing the vitamin

C assumes significance. In view of the widespread use of vitamin C throughout the

globe, a large number of methods have been developed. Accordingly, an attempt has

been made to review these methods as presented here.

1.2.1 Titrimetric methods

Many titrimetric methods using different titrants have been reported which are

discussed briefly below.

Tetrachlorobenzoquinone 18,19

is recommended as titrant for ascorbic acid. The

titration is carried out in presence of EDTA which act as an indicator as well as

masking agent for associated metal ion impurities. The end point is detected by the

appearance of golden yellow color. Mixtures of ascorbic acid with thiols like cysteine,

o-mercaptobenzoic acid, mercaptosuccinic acid and 3-mercaptopropionic acid cannot

be resolved. However, the interferences of thiols is reported to be avoidable by

masking with acrylamide. The use of dihydroxyindole20,21

in the titrimetric

12

determination of ascorbic acid in fresh fruits and in sulphur containing chinese drugs

has also been reported.

2,6 dichloroindolphenol22

has been a popular reagent for the direct titration of

Ascorbic acid. This method is based on the reduction of DCIP with ascorbic acid in

acidic solutions. In the official method, the solution containing about 2 mg of ascorbic

acid and 5ml of a mixture of metaphosphoric acid and acetic acid is titrated with a

standard solution of DCIP. The titrant acts as a self indicator. Though it is an official

USP method23

as well for the determination of ascorbic acid, yet it is not applicable to

many pharmaceutical preparations containing Fe (II), Sn(II), Cu(II), SO2, SO32-

and

S2O32-

ions since higher amounts of ascorbic acid contents are observed in presence of

these ions which are usually associated with mineral or liver preparations. Also, the

substances naturally present in fruits or biological materials such as tannins, betannins,

sulfhydryl compounds are oxidised by the dye. The method is applicable only when

the concentration of DHAA is negligible. DHAA may arise due to the presence of

ascorbic acid oxidase or Fe(III) or Cu(II) ions during the preparation of samples.

However, the formation of DHAA24

during the preparation of plant samples can be

minimized by the addition of trichloroacetic acid. The alkalinity of the same also

hinders the determination. A modified method25

for titrating the citrate buffered

solutions (pH 3.5) with alcoholic solution is proposed for the estimation of ascorbic

acid in pharmaceutical preparations. The alcoholic solution is claimed to be superior to

official solutions mainly because of its stability, but it requires storage in a

refrigerator.

Cerium (IV) sulphate has been used as reagent for the determination of

ascorbic acid. After adding an excess volume of cerium (IV) sulphate solution,

excess of Ce(IV) is back titrated with iron(II) solution only after waiting for 30 min.

Different indicators such as diphenylbenzidine, ferroin, N-phenyl-anthranillic acid

and rhodamine 6G are used but with 2-3% higher consumption of titrant. Rao and

Sastry used ferroin26

as an indicator in 0.75-1.25M sulphuric acid solution containing

phosphoric acid. Other indicator such as perphenazine27

, chloropromazine

hydrochloride28

, methiomeprazine hydrochloride, and methiotrimeprazine maleate29

13

in HCL, H2SO4 or H3PO4 medium have been used for the direct determination of

ascorbic acid with 0.05 M cerium(IV) sulphate and potassium dichromate.

Ferricinium cation30

reacts with ascorbic acid to give a yellow colored extract

in chloroform from solutions of pH 2-3 which shows maximum absorbance at 440 nm.

A linear relationship between absorbance and concentration is observed up to 600μg

of ascorbic acid. The method requires rigid control of pH in the range 2-3 otherwise

the ferricinium cation decomposes to ferrocene and other products in neutral or

slightly acidic medium. Only minute quantities of vitamin B12 and folic acid are

tolerated. Iron(II) does not interfere.

Potassium hexacyanoferrate(III)31,32

has been used by different workers for the

visual titration of ascorbic acid in slightly alkaline medium buffered with sodium

acetate using DCIP as indicator. Sensitivity and accuracy are found to be improved at

pH 8.3-8.4. The titrant has also been used in acid solutions such as 4-5 M sulphuric

acid, 12-14 M acetic acid and 0.4-1.5 M sulphuric acid, using diphenylamine sulfonate

and diphenylamine as indicators respectively.

Recently, a new titrimetric method is reported for the determination of ascorbic

acid with ammonium hexanitratocerate (IV) reagent33

solutionin nitric acid. The

method is used for the determination of ascorbic acid in pure and pharmaceutical

preparations.

Several titrimetric reagents involving iodine/bromine have been used for the

determination of ascorbic acid as summarized in the following:

Determination of ascorbic acid with iodine, potassium iodate34

, potassium

bromate and iodine monochloride using starch as an indicator is reported by many

workers. Some other reagent such as variamine blue in the presence of mercuric

chloride and p-ethoxychrysoidine as indicators have been recommended and their

use in ascorbic acid assay was reviewed by Rao35

, who proposed naphthol blue

black, amaranth or Brilliant Ponceau 5R as alternative indicators. Cu(II), As(III),

Hg(II), cysteine, thiourea, thioglycollic acid, sulphide and sulphite interfere

seriously. The indicators such as perphenazine36

, 1-amino-4-

14

hydroxyanthraquinone37

, azine and oxazine dyes38

phenosafranine and

aposafranine39

, have also been used in the bromatometric estimation of ascorbic acid.

N-bromosuccinimide40

has been used for the determination of ascorbic acid

using starch as an indicator. Reductones, reductic acid and iron salts do not interfere

with this titration. But, it was found to give satisfactory results with samples of

preserved juices and squashes containing metabisulfite as a preservative. However,

bisulfite can be complexed with acetone before carrying out the titration. Evered 41

modified the procedure by extracting the liberated iodine in an organic solvent to

avoid the interference from highly colored samples. Quinolone yellow solution was

used for detecting the end point in titrations with N-bromophthalimide and N-

bromosaccharin42

. Cysteine and glutamic acid are found to be interfere in this

method. Chauhan and singh43

made use of N-bromosaccharin for the micro

determination of ascorbic acid in pure solutions and pharmaceutical preparations.

O-iodosobenzoate44

and o-diacetoxyiodobenzoate45

have been used in the

recent past for the titrimetric determination of vitamin C in different samples at

neutral pH using leuco-2,6-dichlorophenolindophenol plus potassium iodide as an

indicator. Thiourea, thiosulfate, sulphide and thiosemicarbazide interfere seriously.

Cysteine and glutathione interfere but can be masked by cyanoethylation.

Chloramine T46-48

in presence of acidified potassium iodide or potassium

bromide has been proposed for the determination of ascorbic acid. Oxazine dyes49

also act as indicators in these titrations using 1:1 hydrochloride or acetic acid

solutions containing potassium bromide. Some of the organic dye stuffs used as

indicators acts by being destroyed at the equivalence point. N-substituted

perphenazines50

are also used as redox indicators in the titrations with chloramine T

and chloramine B.

Thallim(III) perchlorate51

and copper(II) sulfate52

have been reported for the

analysis of ascorbic acid in acidic medium but the former requires an inert

atmosphere. The ascorbic acid in fruits is estimated indirectly by determining the

unreacted thallium(III) iodometrically.

15

1.2.2 Non spectrophotometric methods

Flourometric methods53-59

have been used for the determination of ascorbic

acid in different samples including fruits, vegetables and human serum. The formation

of fluorescent product by the reaction of DHAA with o-phenylenediamine is the basis

of many such methods53-57

including the AOAC method58

which involves the

oxidation of ascorbic acid with different oxidants such as activated charcoal and dilute

iodine solution. An improved microfluorimetric method using DCIP as an oxidising

agent instead of acid-washed norit, has been reported thus avoiding the filteration and

various transfer operation steps involved. Weiquian56

has recommended the use of a

less expensive oxalic acid-acetic acid solution instead of meta-phosphoric acid-acetic

acid solution for extracting the sample. Dehydroreductic acid, dehydroreductones, and

alloxan are reported to interfere.

Iwata et al60

reported an ultramicro fluorometric determination of ascorbic acid

in human serum by using the reaction between DHAA and 1,2-diamino-4,5-

dimethoxy-benzene. A method based, on oxidation of DHAA with 2-

hydroxynaphthaldehyde thiosemicarbazone in the presence of Mn(II) catalyst or its

activating effect on oxidation of rhodamine 6G by potassium bromate in the presence

of vanadium(V) has been reported in pharmaceuticals and urine by kinetic

fluorometry. A flow injection stopped flow spectrofluorometric determination of total

ascorbic acid (0.025-1.0 μg ml-1

) based on enzyme linked coupled reactions that

produces a fluorescent quinoxaline was proposed by Huang et al61

. A laccase based

micellar enhanced spectrofluorometric analysis and similar enhancing effect on a

mimetic enzyme- catalysed reaction was used for the determination of ascorbic acid

by the same workers62,63

. The photooxidation of ascorbic acid sensitized with thionine

blue was investigated by perez-ruiz et al64

for its determination in pharmaceuticals,

fruit juices and soft drinks. The fluorescence of the system of calcein-copper(II)-

ammonium thiocyanate65

has been used for the determination of ascorbic acid in

vegetables.

A new fluorimetric sensor66

for the determination of ascorbic acid (AA) is

reported. The method is based on complexation between ascorbic acid and silver

16

nanoparticles and the quenched fluorescence intensity was linear with the

concentration of ascorbic acid in the range of 4.1 x 10-6

to 1.0 x 10-4

M with a

detection limit of 1.0 x 10-7

M. The proposed method was applied to the determination

of ascorbic acid in vegetables and vitamin C tablets

Feng et al67

reported a spectrofluorimetric method for the determination of

trace amount of ascorbic acid which is based on the inhibition of ascorbic acid on the

oxidation of pyronine Y (PRY) by nitrite. The detection limit for ascorbic acid is

0.012 μg ml-1

, the linear range of the determination is 0.02-0.36 μg ml-1

. This method

has been used to determine ascorbic acid in pharmaceuticals, vegetables, fruits and

soft drinks.

Wang et al proposed a spectrofluorometric method68

for the determination of

ascorbic acid (AA) based on its activation on the haemoglobin-catalysed reaction. The

fluorescence intensity of the product was measured under the optimal experimental

conditions, i.e. 4.0 x 10-6

M H2O2, 6.0 x 10-5

M p-cresol, 1.2 M NH3-NH4Cl (pH 10.4)

and 2.0 x 10-7

M haemoglobin. The activation of AA was found to be associated with

a high ammonia concentration. The linear range of the method was 9.0 x 10-10

- 3.6 x

10-8

M of AA.

Chemiluminescent methods

Different systems involving Cu(II)-luminol, Ce(IV)-rhodamine 6G, Fe(II)-

luminol-O2, H2O2-luminol-peroxidase, H2O2-hemin-luminol and H2O2-luminol-KIO4

form the basis of many chemiluminescent(CL) methods69-77

. The upper limit of the

linear range varies from 2.0 x 10-6

-6.0 x 10-5

mol L-1

and the detection limit lies in the

range 1.0-6.2 x 10-7

mol L-1

of ascorbic acid. A highly sensitive CL procedure

78 for the

determination of ascorbic acid (1 x 10-9

-1 x10-6

μg ml-1

) in vegetables and tablets with

reversed flow injection analysis (FIA) is reported which is based on the inhibition of

CL reaction of luminol-Fe2+

-O2 system by ascorbic acid.

Pires et al79

reported a multi-pumping flow-based method with

chemiluminescent detection for the determination of ascorbic acid in fruit juices

(powdered form). The method relies on the inhibitory effect of AA on the oxidation of

17

luminol by hydrogen peroxide in alkaline medium. The analytical curve is linear up to

about 11 mmol l-1

of AA.

Recently ,Chen et al80

proposed a method in which a strongly

chemiluminescence (CL) is formed from the mixing of hydrogen peroxide (H2O2),

hydrogen carbonate (HCO3-) and CdSe/ CdS quantum clots and the addition of trace

amount of L-ascorbic acid into the CL system caused quenching of luminescence

intensity. The CL intensity and the concentration of L-ascorbic acid have a good linear

relationship in the ranges of 1.0 x 10-7

-1.0 x 10-4

mol L-1

. This method has been

applied to determine L-ascorbic acid in human serum.

Danet et al81

reported a method for determination of ascorbic acid from fruit

juices by combining a flow injection analysis (FIA) system with a chemiluminometric

detector and a reactor with L-ascorbate oxidase immobilized on controlled pore glass.

It was found that ascorbic acid gives chemiluminescence with luminol in the presence

of hexacyanoferrate (III) in an alkaline solution.. Accordingly, two

chemiluminometric signals were registered for each determination, one signal

corresponding to the sample that passed through the enzymatic reactor that

decomposed the ascorbic acid completely, and the second signal corresponding to the

sample that does not pass through the reactor. The difference between the two signals

corresponds to ascorbic acid from the sample. The linear range of the method was 10-

1000 m molL-1

of ascorbic acid.

Kinetic methods

A few kinetic methods with or without the use of stopped flow technique have

been proposed for the determination of ascorbic acid. These methods generally

involve the reducing effect of ascorbic acid on DCIP82,83

(λmax = 522 nm) or toluidine

blue84,85

(λmax = 600 nm), ammonium molybdate86

(λmax = 800 nm). Similar kinetic

methods were reported either by measuring the decrease in absorbance of Co(III)

complex87

with EDTA or with 5,10,15,20-tetrakis-(4- N-trimethyl-

aminophenyl)porphyrin at 540 nm and 400 nm respectively or using 5-[N-(3,5-

dichloroquinoneimine)]-8-hydroxyquinoline88

in the presence of 20% ethanol and 0.08

18

M britton-robinson buffer (pH 5.5). Zhang et al89

made use of catalytic kinetic

spectrophotometry for determining ascorbic acid contents upto 0.6 μg ml-1

(λmax = 555

nm) in hydrochloric acid-potassium dihydrogenphosphate buffer containing the

rhodamine B, vanadium ion and poyassium bromate solution. The inhibiting effect of

ascorbic acid on the oxalate/ K2Cr2O7-KIO4 or KI/ rhodamine 6G has been used90-92

for its determination.

Another flow-injection kinetic spectrophotometric method90

which is based on

the inhibiting effect of ascorbic acid on the enhancing effect of oxalate on the

potassium dichromate-potassium iodide/ rhodamine 6G system. The detection limit

and linear ranges are 0.08 and 0.10-4.00 μg ml-1

respectively. This method has been

used to determine ascorbic acid in pharmaceuticals, tomatoes and oranges. Recently, a

catalytic kinetic spectrophotometric method91

for the determination of trace amounts

of ascorbic acid has been reported by Zhen-xin, et al. The method is based on the

activation effect of ascorbic acid on vanadium (V) catalyzed oxidation of rhodamine B

by potassium bromate in weak acidic medium at pH 4.5. The absorbance was

measured at 555 nm. The linear range of the method is 0-7.0 μg ml-1

.

Liu et al92

developed a kinetic method performed on a flow injection system

for the determination of ascorbic acid by using its catalytic effect on the complexation

reaction of Cu(II) with 5,10,15,20-tetrakis-(4- N-trimethyl-aminophenyl)porphyrin.

The characteristic spectrum of porphyrin (Sorer band), which shows intense

absorption around 400 nm. By incorporating the complexation reaction into a flow

injection system, ascorbic acid could be determined either over a broad dynamic range

of 0.1-1000 μg ml-1

or at a trace level below 5 μg ml-1

.

Titrimetric FIA93-95

based on the reducing behaviour of ascorbic acid has been

used in the analysis of synthetic samples, urine and soft drinks using chloramine T-

starch or KBr- methyl red as indicators or Ce(IV) as titrant in 0.1 m sulphuric acid

medium as a self indicating system. However the method94

using Ce(IV) as titrant

requires rigid control of acidity. Some of the methods based on the reduction of

iron(III) by ascorbic acid and the measurement of reduced iron (II) by its

complexation with o-phen96-100

or oxidation of ascorbic acid by thalium (I) or a

19

catalysed light emission from luminol101

or and photochemical reduction of methylene

blue102-104

have been reported.

Sequential injection analysis spectrophotometric method105

for the assay of

vitamin C in drug formulations is based on the oxidation reaction of vitamin C with

cerium(IV) in sulfuric acid media using a spectrophotometer as a detector with the

wavelength monitored at 410 nm. A linear calibration plot for the determination of

vitamin C was obtained in the concentration range 30 - 200 ppm.

Electrochemical methods

Voltametric analysis using different electrodes like conventional

electrodes106,107

microdisc electrodes108

, carbon paste electrodes109-112

, microband

and multiple band electrodes113

and ferrocene modified114

green pepper seed carbon

paste electrode, boron- doped diamond electrode using cyclic voltametry115

and

differential pulse voltametry116

have been used in the determination of ascorbic acid.

However, the use of such electrodes is not so much reliable because of electrode

fouling by oxidation products.

Electrocatalytic oxidation of ascorbic acid has been carried out on different

modified electrodes such as Co-Salen polymer electrode117

, ferrocene, β-cyclodextrin-

ferrocene, inclusion complex-carbon paste electrodes118,119

. Quantitation of ascorbic

acid has also been carried out by stripping voltametry120

on a glassy electrode and by

cyclic voltametry using ruthenium dioxide counter electrode121

or by means of

electrostatically trapping Mo(CN)84-

mediator in the cationic film of glutaraldehyde-

cross-linked poly-L-lysine122

. Ascorbic acid has been determined by differential pulse

polarogrphy123-127

after derivitization with o-phenylenediamine.

Amperometric analysis has also been used for the determination of ascorbic

acid. These methods generally involve sensor‟s128-132

which are basically constructed

by immobilizing ascorbate oxidase in the reconstituted collagen membrane or

glutraldehyde and mounting the product on a clark oxygen electrode. Enzymeless

amperometric biosensor‟s133

for L-ascorbate has been prepared by entrapping poly-L-

histidine in polyacrylamide gel and its treatment with cupric chloride.

20

An amperometric biosensor method132

based on the determination of the

decrease in the dissolved oxygen level. The zucchini (Cucurbita pepo) tissue

homogenate was crosslinked with gelatine using glutaraldehyde and fixed on a

pretreated teflon membrane. The zucchini tissue contained the enzyme ascorbate

oxidase and this enzyme catalyzed the oxidation of ascorbic acid in the presence of

dissolved oxygen,which results a decrease in the dissolved oxygen related to ascorbic

acid concentration. A linear response was observed from 5x10-6

M to 1.2x10-3

M for

ascorbic acid.Munoz et al134

reported amperometric method for quantification of

ascorbic acid (AA) in pharmaceutical formulations using flow-injection analysis

(FIA). A slice of recordable compact disc (CD) modified by electrodeposition of

platinum was employed as the working electrode. A. De Donato et al135

made use of a

sessile mercury drop electrode for the determination of ascorbic acid.

Potentiometric titrations136-141

have been employed for the determination of

ascorbic acid using DCIP, copper sulphate, N-bromosuccinimide, iodine, potassium

hexacyanoferrate and tetrachlorobenzoquinone as titrants. Microdetermination of L-

ascorbic acid based on its oxidation with iodine in chloroform137

or methanol140

has

also been carried out, which involves the determination of iodide ion formed, with

iodide ion selective electrode142

. Some potentiometric methods using the electrodes

such as carbon paste electrode143

, modified carbon paste electrodes144

, graphite carbon

electrode145

or a derivative of Co(II)-phthalocyanine doped with iodine146

and

immobilized enzymic (ascorbate oxidase) electrodes147,148

have been reported for the

assay of ascorbic acid.

Fernandes et al148

reported a potentiometric sensor for L-ascorbic acid based

on the redox properties of copper(II) ions incorporated in a EVA membrane. The

poly(ethylene-co-vinyl acetate) matrix was doped with Cu2+

ions and dispersed on the

surface of a graphite/ epoxy electrode. The electrode displayed a Nernstian response in

the ascorbate concentration between 5.6 x 10-6

and 3.7 x 10-4

mol L-1

, in the presence

of 0.1 mol L-1

KH2PO4 buffer at pH 5.0 and at 250C.

21

Paim et al142

reported a potentiometric flow titration for the determination of

ascorbic acid in pharmaceutical formulations. The method is based on the reduction of

IO3- by ascorbic acid and the detection was carried out employing a flow-through ion

selective electrode for iodide. The titration system allowed the determination of

ascorbic acid in pharmaceutical formulations with concentrations ranging from 7.5 to

15.0 m mol l-1

.

1.2.3 Miscellaneous methods

The techniques, C13

NMR, PMR spectroscopy149-151

, ESR spectroscopy152-154

,

FT-IR spectrometry155

, Near-infrared reflectance spectroscopy156

, electrospray

ionization tandem mass spectrometry (ESI-MS)157

have been shown to be useful in the

determination of ascorbic acid. The use of other techniques such as differential158-160

or first/ second/ third-order derivative or double divisor ratio spectra derivative

spectrophotometry158,161-165

or solid phase spectrophotometry166-168

or diffuse

reflectance UV-visible absorption spectrometry169

or UV spectrophotometry using

factor analysis (PARFAC) and partial least square (PLS) method170

have been reported

for direct determination of ascorbic acid in the range 200-320 nm in soft drinks and

other products.

Recently, a method171

for the determination of ascorbic acid based on the

formation of K2Zn3[Fe(CN)6]2 nanoparticles by resonance Rayleigh scattering , which

involves potassium ferricyanide (K3[Fe(CN)6]) reaction with ascorbic acid to produce

potassium ferrocyanide K4[Fe(CN)6] , which further reacted with Zn2+

to form

potassium zinc hexacyanoferate K2Zn3[Fe(CN)6]2 nanoparticles in Britton-Robinson

buffer medium (pH 4.43). It was found that the RSS intensity of the system at the RRS

peak of 363.4 nm was proportional to the ascorbic acid concentration in the range of

4.0-80.0 mol L-1

, and the detection limit for ascorbic acid was 0.075 mol L-1

.

Atomic absorption spectrometry172-174

has also been used for the determination

of ascorbic acid. An indirect method172

based on the reduction of chromium(VI) to

chromium(III) with the ascorbic acid and separation of unreacted Cr(VI) as its 1,5-

22

diphenylcarbazide complex on a column filled with Amberlite XAD-16, elution of the

complex by 10 ml of 0.05 mol l-1

H2SO4 in methanol, and determination by flame

atomic absorption spectrometry is reported. Amount of the ascorbic acid is calculated

from the amount of Cr(VI) reacted with ascorbic acid. This method allows

determination of ascorbic acid in the range 0.5-20 μg ml-1

. A flow-injection flame

atomic absorption spectrometry173

for indirect determination of ascorbic acid based on

its reducing action to Fe(III) is reported. The Fe(II), produced by the reduction of

Fe(III), is on-line adsorbed on a mini-column filled with cation-exchange resin to

concentrate, then eluted reversely by 3 mol l-1

nitric acid to nebulizer and measured

by flame atomic absorption spectrometry, while the excessive Fe(II) reacts with NH4F

to form anion [FeF6]3-.

The absorbance of Fe(II) is proportional to the concentration of

ascorbic acid in the range from 0.1 to 50 mg l-1

.

A similar method174

based on the redox reaction between chromate and

ascorbic acid in acid medium is reported. The Cr(III), thus reduced by ascorbic acid, is

adsorbed on a cation exchange resin micro-column, then eluted by 3 mol l-1

nitric acid

to the nebulizer and measured by atomic absorption spectrometry. The absorbance of

Cr(III) is proportional to the concentration of ascorbic acid in the sample. When the

reaction and adsorption time reaches two min, a calibration graph ranging from 0.3 -

60 μg ml-1

of ascorbic acid.

1.2.4 Spectrophotometric methods

Several dyes such as dimethoxyquinone (DMDQ), ninhydrin 2,6-

dichloroindolphenol (DCIP), fast red AL salt and 2‟,7‟-dichlorfluorescein etc. have

been used for the determination of vitamin C. Among these dyes, DCIP has been most

extensively studied. It is included in the official titrimetric methods as reported in

different pharmacopoeias175-177

and it also forms the basis of many colorimetric

methods. The blue dye DCIP is reduced to the colorless form on addition of ascorbic

acid, but it gives a pink color to the acidic solutions. Using the dye, ascorbic acid

present in human urine178

and processed potatoes179

has been determined. The excess

23

dye can be extracted with xylene or butanol. Many substances which are capable of

reducing the dye resulting from the preparation and processing of food sample

interfere. Flow injection analysis proposed by Gary et al180

and continuous flow

systems have been used to monitor the decrease in absorbance of DCIP. Such

automated systems appear to be justified only when routine analysis of a large number

of samples is needed, otherwise it is tedious to use for a single estimation.

Ninhydrin181,182

(1) and methylene blue183

(2) find applications with the

determination of ascorbic acid in food products. . The reaction of ascorbic acid with

ninhydrin carried out on a water bath using 80% aqueous solution as a medium in

0.01M NH4OH is used for its determination in pharmaceuticals (λmax = 415 nm). The

colorless form of dye (2) is extracted into chloroform after its reduction with ascorbic

acid; back oxidation of the dihydro derivative to methylene blue has been used for the

assay of ascorbic acid (λmax = 653 nm). The method is reported to be highly sensitive.

Dimethoxyquinone184

gives a violet-colored product with ascorbic in phosphate

buffer (pH 6.6). The reduced „indigoid‟ form is perhaps responsible for the formation

of violet-colored solution which is stable over 24 hrs. only under dark conditions is

measured at 510 nm. Beer‟s law holds good up to 80 μg ml-1

with a detection limit of

10 μg ml-1

. Riboflavin and copper interfere. The interference of iron (II) sulphate

responsible for precipitation can be removed by centrifugation. Though the method is

not sufficiently sensitive (ɛ = 1.62 103), it can still be applied to the analysis of citrus

fruits185

after extracting the colored product into chloroform (λmax = 530 nm). Lin et

al186

, and Pandey187

reported procedures based on the reaction of ascorbic acid with

fast Red AL salt (3) (zinc chloride salt of diazotized 1-aminoanthraquinone), and

tetrachlorobenzoquinone (4). The reaction of reagent (3) proceeds in acidic medium

but the blue color develops only after addition of alkali, which exhibits three

absorption bands in between 500-630. If one uses the later reagent (4), ascorbic acid is

determined at 336 nm via a decrease in absorbance of 7 10-4

m

tetrachlorobenzoquinone (chloranil) in 80% acetone-water (v/v) medium. With these

methods, mixtures of ascorbic acid with thiols like o-mercaptobenzoic acid,

mercaptosuccinic acid, 3-mercaptopropionic acid cannot be resolved.

24

A spectrophotometric method has been developed for the determination of

ascorbic acid which reduces methyl viologen188

to form a stable blue coloured ion.

This method has a sensitivity and lower limit detection of 0.1 μg ml−1

of ascorbic acid

(0.1 ppm). Beer‟s law is obeyed over the concentration range of 1.0–10 μg ml−1

of

ascorbic acid per 10 ml of the final solution (0.1–1.0 μg ml−1

) at 600 nm.

Ascorbic acid reacts with potassium iodide-iodate solution under acidic

conditions (pH 4.0-4.8) to liberate iodine and the liberated iodine selectively oxidizes

leucomalachite green189

(LMG) to malachite green (MG) dye. The colour of the dye is

measured at 620 nm. Beer‟s law is obeyed over the concentration range of 0.8-8 μg of

AA/ 25mL of final solution (0.032-0.32 ppm). The bleaching action of liberated

iodine190

on Rhodamine B and auramine-O dyes has also been used for the

determination of ascorbic acid. The liberated iodine bleaches the pinkish red colour of

the Rhodamine – B dye191

. The colour of the dye is measured at 555 nm.

A new method (A) based on the oxidation of ascorbic acid (AA) by known

excess of Se(IV) in hydrochloric acid medium and subsequent determination of

unreacted Se(IV) by reacting it with iodide in the same acid medium to liberate iodine,

which react with starch to form a stable blue coloured iodine-starch complex, which

shows maximum absorbance at 590 nm is proposed192

. Another method (B) based on

the oxidation of ascorbic acid (AA) by known excess of Cr(VI) in sulphuric acid

medium and the determination of unreacted Cr(VI) with diphenyl carbazide (DPC)

under the same acidic medium to produce a stable red-violet coloured species, which

shows a maximum absorbance at 550 nm. The apparent molar absorptivity values are

found to be 1.627x104 and 1.641x10

4 l mol

-1 cm

-1 for methods A and B respectively.

Some methods involving the coinage metals (Cu, Ag) complexes have been

worked out. The reduction of Cu(II) in a biphasic system of isoamyl alcohol and an

aqueous solution of pH 4.6 to Cu(I) , followed by its complexation with cuproine to

give a red colored complex (λmax = 454 nm),was reported by Contreras et al193

for the

analysis of foods and vegetables. Fresh fruits and vegetables and dehydrated samples

were analysed after extracting with 5% HPO3 and with a 1:1 mixture of 0.5% HPO3

and 0.1 M H2SO4 respectively. Also the colored complexes of Cu(I) with 2,2-

25

biquinoline194

(λmax = 540 nm), rhodanine195

(λmax = 473 nm), and 2,9-dimethyl-1,10-

phenanthroline190-200

(λmax = 450 nm) have been used to determine ascorbic acid in

different samples.

A modified method199

for ascorbic acid determination is based on the oxidation

of AA to dehydroascorbic acid with a Cu(lI)-2,9-dimethyl-1, 10-phenanthroline

[neocuproine (Nc)] reagent in ammonium acetate-containing medium at pH 7, where

the absorbance of the formed bis (Nc)-copper(l)chelate is measured at 450 nm. This

chelate is formed immediately and the apparent molar absorptivity for AA is found to

be 1.60 x 104 dm

3 mol

-1 cm

-1. Beer ' s law is obeyed within concentration range of 8.0

x 10-6

and 8.0 x 10-5

M. The method is applied to a number of commercial fruit juices,

pharmaceutical preparations containing Vitamin C, and red wine.

The use of Cu (II) as vitamin C oxidant has been utilized for its

determination200

. After completion of oxidation reaction, excess of Cu (II) is

determined by complexation with alizarin red‟s (ARS). Thiocyanate ion is used as

stabilising agent for Cu (I) which is the product of oxidation reaction. This method is

used for determination of vitamin C in fruits and pharmaceutical products. Linearity

was found to be in the range 3.5 x 10-6

- 4.8 x 10-5

M (0.6-8.2 ppm). The decrease in

absorbance (λmax = 600 nm) of copper(II)-ammonia complex by the addition of

ascorbic acid has been used for its determination in pharmaceutical preparations201

.

During this reaction, ascorbic acid is oxidized and the copper(II)-ammonia complex is

reduced to the copper(I)-ammonia complex by the addition of ascorbic acid.

A similar type of reaction was used by Zarei et al202,203

where standard

addition method is applied for simultaneous determination of citric and ascorbic acid

or selective determination of ascorbic acid in presence of citric acid. The method is

based on the difference in the rate of reaction of citric and ascorbic acid with

copper(II)-ammonia complex. A new spectrophotometric method for the

determination of ascorbic acid using Ag(I) is based on photochemical reaction204

between ascorbic acid and Ag(I) in aqueous triton X-100 medium was used for

ascorbic acid in pharmaceuticals. Ag(I) was photochemically reduced by ascorbic acid

to yellow silver having particle with nm range size which shows absorption at 415 nm.

26

Analytical applications of molybdenum blue formed on reduction of

phosphomolybdate complex205

, ammonium molybdate206-208

or molybdic acid209

have

been reported by many workers for the determination of ascorbic acid in

pharmaceuticals, fruits and vegetables, pastries and bevereges. Ammonium

molybdate-sulfuric acid system requires 1 hr. for complete development of color with

ascorbic acid. However, such waiting time can be decreased to 15 min by the addition

of metaphosphoric acid-acetic acid solution207

. The colored species obeys Beer‟s law

over the range 2-32 μg ml-1

at 760 nm. Serious interferences are observed due to

phenolic compounds such as catechins, gallic acid, pyrogallol and gallotanins,

thiosulfate ions and thiourea. Molybdenum isopoly-acid210

has been used to produce

heteropoly blue for the assay of ascorbic acid over the range 0-12 mg/ lit. Another

method211

is based on the reduction of phosphomolybdic acid by ascorbic acid to

molybdenum blue. The reduced product is monitored at 744 nm. In yet another

method212

where electrochemical reduction of the 18-molybdo-2-phosphate heteropoly

anion by ascorbic acid depending on the ratio of concentration, of the reagent and the

reducing agent (i.e. ascorbate). Two-electron heteropoly blue (in the excess of the

reagent, λmax = 790 nm, Ԑ = 1.17 × 104 l mol

-1 cm

-1) or four-electron heteropoly blue

(in the excess of the ascorbic acid, λmax = 680 nm, Ԑ = 2.16 × 104 Lmol

-1cm

-1) are

formed. The detection limit of method is 2.4 10-7

mol l-1

. The use of folin

reagent213,214

and folin phenol215

(λmax = 760 nm) has also been described for the assay

of biological samples after deproteinizing with TCA. Beer‟s law is obeyed upto 45 μg

ml-1

. The color development is not obstructed by bovine serum albumin, adenine,

guanine, thymol and oxyhaemoglobin. Folin-ciocalteu216

reagent reacts with ascorbic

acid to give a blue colored complex (λmax = 730 nm) as well. However the method is

time consuming, as the full color development requires 40-50 min. Ammonium

metavanadate217

gives a green color (λmax = 680 nm) on heating for 10 min in the

presence of ascorbic acid. Though the method has been put to use for the analysis of

some samples, it is not sufficiently sensitive. The activation effect of ascorbic acid on

vanadium (V) catalyzed oxidation of deoxidized rhodamine B by potassium bromate

in weak acidic medium at pH 4.5 has been used for the determination of ascorbic

27

acid218

. The absorbance is measured at 555 nm. The linear range of the method was 0-

7.0 μg ml-1

. The detection limit for ascorbic acid was 2.5 x 10 -7

g l-1

.

Hashmi et al219

proposed a method based on the reaction of 2,3,5-

triphenyltetrazolium chloride with ascorbic acid in alkaline medium. The pink solution

is allowed to stand in the dark for 30 min. at 25oC, it obeys Beer‟s law over the range

5-25 μg ml-1

. Riboflavin, cyanocobalamin and folic acid interfere due to their own

color. A similar but modified method220

is developed for the determination of ascorbic

acid after extracting the pink colored species (λmax = 480 nm) formed by the

interaction of ascorbic acid and 2, 3, 5-triphenyltetrazolium chloride into chloroform.

Beer's law holds well over the range 5.0-40.0 μg ml-1

of ascorbic acid. The method is

used in the analysis of different pharmaceutical products. Other derivatives such as

2,5-diphenyl-3-thiazolyl-tetrazolium chloride221

at pH 12.2, 2-(p-iodophenyl)-3-(p-

nitrophenyl)-5-phenyltetrazolium chloride at pH 10.5 (λmax = 540 nm) and 2,2‟,5,5‟-

tetra-(4-nitrophenyl)3,3‟-(3,3‟-dimethoxy-4,4‟-biphenyl) ditetrazolium chloride222

have also been employed for assay of ascorbic acid. M. Yang et al223,224

have proposed

the use of 2-[2-(6-methylbenzothiazolyl)azo]-5-diethylamino benzoic acid in acetic

acid-sodium acetate buffer (pH 5.8) and 4-[2-benzothiazolylazo)]-pyrocatechol for the

determination of ascorbic acid in pharmaceuticals by indirect spectrophotometry.

The reaction of hexacyanoferrate (III)225

was used for the determination of

micro quantities of vitamin C by measuring the decrease in color intensity of the

reagent (λmax = 420 nm) in Mcllvaine buffer (pH 5.2) solutions. Beer‟s law is restricted

within the range 180-270 μg of ascorbic acid. In general, all such reagents that reduce

hexacyanoferrate (III) or oxidise hexacyanoferrate (II) under experimental conditions

interfere. Further the utility of the method is limited to colorless solutions. The same

procedure has been used for the determination of vitamin C content in grapes

successfully226

. Recently, Bead Injection Spectroscopy-Flow Injection Analysis (BIS-

FIA) system227

with spectrophotometric detection is used by decrease in absorbance

(λmax = 720 nm) when Prussian blue (PB) is reduced by ascorbic acid. The

chromogenic reagent (PB) is injected into the carrier and immobilized on beads. When

sample is injected, reaching the bead surface where PB is sorbed, ascorbic acid

28

converts it to Prussian white form, which is transparent, producing the discoloration of

the detection zone. At the end of the analysis, beads are discarded by reversing the

flow and instantaneously transported out of the system. The calibration graph was

linear over the range 5.1 x10-6

-6.8 x 10-5

M. By using Flow injection analysis228

,

ascorbic acid can also be determined with the same reaction involved. This reaction

was evaluated for spectrophotometric determination of ascorbic acid employing

Fe(III) and hexacyanoferrate(III) as chromogenic reagents. An excess of the

complexing anion avoids the formation of ppt. and forms a deep blue soln. when

Fe(III) is reduced to Fe(II) by ascorbic acid. The max absorbance of the colored

complex occurs at 700 nm and the molar absorptivity is 3.0×104 l mol

-1 cm

-1.

Many spectrophotometric methods based on the reduction of Fe(III) to Fe(II)

with ascorbic acid, followed by the complexation of reduced Fe(II) with different

reagents viz. α,α‟-bipyridyl229-237

, 1,10-phenanthroline238-48

, oximes249-251

,

oximinocyclohexanone252

have been reported. Amongst them, α,α‟-bipyridyl and 1,10-

phenanthroline find extensive use in the development of analytical procedures. Most

of these methods are time consuming, as full color development is achieved only after

waiting for 30-60 min. Micro modification of the procedure applicable to human

plasma and animal tissue has been reported without the interference of glucose,

fructose, sucrose, glutathione and cysteine. The procedure has been simplified by Arya

and Mahajan232

which requires only 5 min, waiting time, instead of 30 or 60 min, with

Beer‟s law ranges up to 12 μg ml-1

(λmax = 522 nm). Total ascorbic acid has been

determined in blood plasma after reducing DHAA with dithiothreitol at pH 6.5-8.0,

removing the excess of dithiothreitol with N-ethylmaleimide and in urine by

acidifying with TCA and shaking with activated charcoal. The reduced Fe(II) forms a

water-soluble colored complex with o-phen. (λmax = 510-515 nm) at 1.5-6.5, with

obedience of Beer‟s law up to 8μg ml-1

. Ascorbic acid in fruits is determined after

extracting the ternary complex of Fe(II) with α,α‟-bipyridyl/ o-phen and

sulfophthalein253

dyes into chloroform (λmax = 602 nm). Ascorbic acid in medicine was

determined by fading spectrophotometric method based on reduction of Fe(III) to

Fe(II) by ascorbic acid and reacting with sulfosalicylic acid.

29

Color forming reactions of Fe(II) with ferrozine254-256

(λmax = 562 nm), quinaldic

acid in presence of pyridine257

(λmax = 380 nm), TPTZ258-260

(λmax = 593, 595 nm) ,

picolinic acid in presence of pyridine261

(λmax= 400 nm) and nitroso salt262

(λmax = 705

nm ) have been used for the determination of vitamin C in a variety of samples. The

reagents picolinic acid and quinaldic acid, when complexed with iron (II) in the

presence of pyridine, resulted in methods used successfully in the analysis of

pharmaceuticals, food products and biological samples. The respective colored

complexes getting extracted into chloroform obey Beer‟s law in the range 0.4-5.6 μg

ml-1

and 2.5-25 μg ml-1

ascorbic acid without the interferences of common ingredients

of the samples studied.

Arya et al263

proposed an extractive spectrophotometric method for the

determination of micro amounts of ascorbic acid. 1-[Thenoyl- (2')-3,3,3-trifluoro-

acetone] (HTTA) reacts with iron (III) in strongly acidic medium giving a red

coloured complex. The method is based on the proportionate decrease in the colour

intensity of Fe(III)-HTTA complex with the addition of ascorbic acid at 405 nm.

Linear calibration curve is observed in the range 1.0-5.0 μg ml-1

of ascorbic acid.

Another method264

involves the reduction of iron(III) to iron(II) and complexation of

iron(II) with 4-(2-pyridylazo)resorcinol, followed by its extraction into n-butanol. The

absorbance is measured at 710 nm. Beer‟s law is observed up to 5.5 μg ml−1

ascorbic

acid.

The reaction of Br-PADAP265

[2-(5-bromo-2-pyridylazo)-5-

diethylaminophenol)] with iron(II) in presence of Triton-X-100 forms a brown

complex with an absorption maxima at 560 and 748 nm and a composition of 1:2

iron(II)-Br-PADAP. The complex is formed immediately and is stable for at least 24

h. The Br-PADAP reagent has an absorption maximum at 487 nm (at pH 4.75). The

reagent and its iron(II) complex have a low solubility in water but the reaction in

presence of Triton-X-100 solves this problem.

Enzymic266-270

colorimetric determinations of ascorbic acid in commercial

vitamin C tablets and in fruits and vegetables were made by measuring the absorbance

at 358 nm or 320 nm of the resulting products obtained by oxidation of o-

30

phenylenediamine/ 1,4-diaminobenzene using ascorbate oxidase or peroxidases in

presence of H2O2 at pH 5.3. Ascorbic acid is determined after oxidation with mercuric

chloride and condensing the DHAA with 4,5-disubstituted phenylenediamine271

,

which gives the quinoxaline derivative used for absorbance measurement. The method

involving 4-nitro-1,2-phenylenediamine272

(λmax = 375 nm) is very complex and

laborious, since it involves many time consuming steps including purification of the

sample with anionic sephadex column.

31

1.3 General Remarks

A large number of methods including titrimetric, spectrophotometric,

instrument based non-spectrophotometric have been reported for the determination of

ascorbic acid. A brief account is of the titrimetric methods given under section (1.2.1).

These methods generally are easy to use, yet some difficulties are noticed with

commonly used titrants such as DCIP, Ceric sulphate or iodine as reported in the

official methods of BP, IP and USP. For instance, in the titration with iodine, it reacts

with other substances other than ascorbic acid such as glutathione, because of relative

strong oxidising nature, thereby restricted its use mainly to pure solutions of ascorbic

acid. Similarly, in the titration with DCIP substances like cysteine and glutathione

interfere. In addition to that, these titrations cannot be used in case of colored samples

or in the presence of reducing substances which can bleach the dye and make the

analysis nonspecific.

There are a number of instrument based non- spectrophotometric methods

(sec 1.2.2) including the techniques such as fluorometry, polarography, amperometry,

enzymatic spectrometric methods. Many electrochemical methods are utilised for the

determination of ascorbic acid but they have not been very popular due to different

experimental problems such as inconvenience of the DME, tendency of the electrodes

to become fouled by adsorption of some substances present in biological samples or

other organic solutes and a relatively poor resolution of such methods in

distinguishing between easily oxidised substances. Further polarographic methods

suffer interferences due to electrochemical impurities present in various ascorbic acid

samples. Amperometric sensors are quite sensitive but have a limited life time. In

coulometry, the requirement for electrode reaction to proceed with 100% efficiency,

and for the stoichiometric and rapid reaction of iodine/bromine generated with

ascorbic acid, is not always met. Chemiluminescence methods are potentially sensitive

but their application requires the modified spectrophotometer. Moreover, lack of

selectivity limits their direct application to real samples. The catalytic methods are

generally more sensitive, but they require both reproducibility of mixing of the analyte

with the reagent and a definite reaction time. Fluorometric methods do not lead to

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direct determination of ascorbic acid instead require calculations involving the

correction of peak heights of blanks, also rigid control of pH is required. Most of the

above mentioned instrument based methods require costly equipment and additional

operator‟s attention, which prevents their applications in small industrial units and

laboratories.

1.4 Aim and Objective of the Work

Vitamin C, also called ascorbic acid, is a powerful water soluble antioxidant

that is vital for the growth and maintenance of all body tissues. So, a daily intake of

vitamin C either in the form of fruits and vegetables or in the form of foods containing

the vitamin is considered necessary for human beings which has resulted a continuous

interest for working out simple and better methods for its determination. A large

number of methods based on different instrumental techniques such as fluorometry,

polarography, CL, spectrometry, flow injection methods and enzymic methods etc. are

available in literature (sec 1.2). But the spectrophotometric methods are preferred for

the routine analysis of vitamin C because of their rapidity and simplicity. These

methods have been developed based on either redox property of ascorbic acid or its

ability to couple with diazotized aniline derivatives to yield colored complexes.

Although many colorimetric methods are reported (sec 1.2.4) but these methods are

associated with their own limitations which require either pretreatment, or lack of

selectivity and sensitivity and many of them are time consuming, thereby restricting

their suitability for frequent use. Keeping in view of these problems, there is much

scope for improvement or devising new methods for the determination of ascorbic

acid. Therefore, the objective of present work is to develop new fast, facile, sensitive

and selective methods adaptable to routine analysis of vitamin C.