A New Spectrophotometric Method for the Determination of Ascorbic Acid

Using Leuco Malachite Green

Kishore K. Tiwari

Department of Chemistry & Biochemistry, Government College of Science, Raipur-492010 (C.G.), India

A new simple and sensitive and selective spectrophotometric method has been developed for the de-

termination of ascorbic acid (AA) at trace level using a new reagent, leuco malachite green (LMG). AA re-

acts with potassium iodide-iodate solution under acidic conditions to liberate iodine and the liberated io-

dine selectively oxidizes LMG to MG dye. The colour of the dye was measured at 620 nm. Beer’s law is

obeyed over the concentration range of 0.8-8 �g AA per 25 mL of final solution (0.032-0.32 ppm). The ap-

parent molar absorptivity and Sandell’s sensitivity of the method were found to be 2.98 � 105 l mol-1cm-1,

0.0042 �g cm-2, and respectively. Statistical treatment of the experimental results indicates that the

method is precise and accurate. The method is free from interference of common ions and many of the in-

gredients commonly found in pharmaceuticals. The reliability of the method was established by parallel

determination against Leucocrystal violet (LCV) method. The method described was satisfactorily ap-

plied for the determination of AA in fruit juices, pharmaceuticals and biological samples.

Keywords: Spectrophotometric method; Ascorbic acid (AA); Leuco malachite green (LMG);

Malachite green (MG); Pharmaceutical and biological samples.

INTRODUCTION

Ascorbic acid is an important vitamin that partici-

pates in wide variety of biological events concerning elec-

tron transport reactions, hydroxylation, and the oxidative

catabolism of aromatic amino acids and so on. It is also es-

sential vitamin for both pharmaceutical and food process-

ing industries. In view of its nutritional significance, varied

uses in food and high daily-recommended doses for hu-

mans. Vitamin C is a very important agent for better public

health.1,2 It is essential for the formation of intracellular ce-

ment substances in a variety of tissue, needed for tissue me-

tabolism, healing of wounds and fractures of bones prevent

scurvy and facilitates absorption of iron.3,4 It has been re-

ported that large doses of vitamin C increases greatly the

rate of production of lymphocytes under antigenic stimula-

tion and it is well established that such a high rate of lym-

phocyte blastomogenesis is associated with a favorable

prognosis of cancer.5 AA is a rather unstable compound;

its content is partially decreasing during food – process-

ing or storage. Because of that, in order to improve nutri-

tive value and to maintain natural properties, vitamin C is

usually added in controlled concentration during the pro-

cessing.6

Many analytical techniques are available for its deter-

mination in different matrices and at different levels. These

techniques include HPLC,7 AFSD,8 voltammetry,9 biosen-

sor method,10 NMR spectroscopy,11 fluorometry,12 enzy-

matic method,13 etc. A number of methods have also been

reported for the spectrophotometric determination of AA.

Among them is the formation of an osazone [bis-(2,4-di-

nitrophenyl hydrazone)] derivative of AA;14 this procedure

is complex, time consuming and subject to several interfer-

ences. Recently modifications have suggested improving

this method.15 The method using 2,6-dichlorophenol indo-

phenol sodium (DCPIP),16 is subject to several limitations.

The silver-gelatin complex has been used in reductive spec-

trophotometric method.17 The oxidation of AA with the

Fe(III) and complexation of resulting Fe(II) with 1,10-

phenonthroline.18 A simple kinetic spectrophotometric

measurement of AA based on the reduction of toluidine

blue.19 An indirect spectrophotometric determination of

AA based on extraction of iodine produced by reduction of

potassium iodate20 has also been reported. Many other re-

agents such as; fast red,21 leucocrystal violet,22 methyl

viologen,23 and rhodamine-B,24 etc are also used in the de-

termination of AA in the past. A few of them are sensitive

Journal of the Chinese Chemical Society, 2010, 57, 105-110 105

* Corresponding author. E-mail: kishoretiwari2003@yahoo.com; kktiwari05@rediffmail.com

but involve costly and carcinogenic reagent while some are

suffering from serious interference. The need for a simple,

sensitive and reliable method for the determination of AA

is clearly recognized.

The aim of present investigation is to demonstrate a

simple and sensitive method suitable for the determination

of AA using LMG as chromogenic reagent. The method has

been successfully applied for the determination of AA in

fruit juices, pharmaceutical and biological samples.

EXPERIMENTAL SECTION

Apparatus and Reagent

A Systronic UV – VIS spectrophotometer 108 with 2

cm matched silica cells were used for all spectral measure-

ments. pH meter model 331 was used for pH measure-

ments. A Remi C-854/4 clinical centrifugal having a maxi-

mum centrifugal force of 1850 gm with fixed swing out ro-

tors was used for centrifugation.

All chemicals used were of AnalaR grade. Double

distilled deionized water was used throughout the study.

AA (Loba Chemie): A stock solution of 1000 �g mL-1

was prepared by dissolving 100 mg of AA in 100 mL of wa-

ter. Working standard solutions were freshly prepared by

appropriate dilution of the stock solutions with water. Po-

tassium iodide (Merck): 0.1-mol l-1 aqueous solution. Po-

tassium iodate (Merck): 0.2-mol l-1 aqueous solution. Po-

tassium iodide-Potassium iodate Mixture: Prepared by

mixing 0.1-mol l-1 potassium iodide and 0.2-mol l-1 potas-

sium iodate in 5:1 ratio. This solution was prepared fresh

daily and kept in amber coloured bottle. Hydrochloric acid:

0.02-mol l-1 aqueous solution was used.

LMG (Sigma-Aldrich, S. Germany): 0.05% solution

was prepared by dissolving 25 mg of LMG {4-((4-(dimeth-

ylamino)phenyl)(phenyl)methyl)-N,N-dimethyl benzene-

amine}, 100 mL of water and 1.5 mL of 85% of phosphoric

acid in a 500 mL of volumetric flask and by shaking gently

until the dye dissolved (phosphoric acid was added to dis-

solve the dye completely and to keep the solution stable for

longer time). The content of the flask were then diluted to

500 mL with water. Acetate buffer25 (pH-(4.5): was pre-

pared by dissolving 13.6 g (1 M) sodium acetate trihydrate

in 80 mL of water, solution pH was adjusted to 4.5 with ace-

tic acid, and the mixture was diluted to 100 mL with water.

Oxalic acid: 0.2-mol l-1 aqueous solution. Sodium salt of

EDTA: 5% aqueous solution, Metaphosphoric acid 3%

aqueous solution.

PROCEDURE

Preparation of calibration graph

An aliquot of sample solution containing 0.8-8.0 �g

AA was transferred in to a series of 25 mL graduated tube.

To this 0.4 mL of potassium iodide-potassium iodate mix-

ture solution and 1 mL of 0.02-mol l-1 hydrochloric acid so-

lution were added, and the mixture was gently shaken until

the appearance of yellow colour, indicating the liberation

of iodine. Then 1 mL of 0.05% LMG solution was added to

it followed by addition of 2 mL of acetate buffer (pH-4.5).

The contents were heated (~ 40°C) in a water bath for 5

min, cooled to room temp and diluted to the mark with dis-

tilled water. The mixture was kept for 10 min for comple-

tion of the reaction. The absorbance of the formed dye was

measured at 620 nm against the reagent blank. The concen-

tration of AA content was established from the calibration

graph.

Determination of AA in pharmaceuticals

All drug samples tested were fresh and purchased

from local pharmacy. An AA tablet or the content of a cap-

sule were weighed, ground in to a fine power and stirred for

2-3 min with 50 mL of deionized water. 1 mL of 5% EDTA

was added and filtered through Whatman No. 41 filter pa-

per. The insoluble mass was washed with three successive

5 mL portions of water and the filtrate plus washings were

diluted to volume in a 250 mL calibrated flask. A known

volume was further diluted depending on the AA content

and the colour of the sample. 1 mL aliquot was analyzed as

recommended above.

Determination of AA in fruit juices

Various samples of fruits like orange, lemon (5 g

each) was weighed, and the juice was separated from the

fruits with a mechanical press and centrifuged in order to

clarify it. A 1 mL aliquot of the juice was diluted to 100 mL

with 0.2 mol l-1 oxalic acid in order to avoid losses of ascor-

bic acid due to air oxidation, 1 mL of 5% EDTA was added

and the solution was centrifuged at 1850 g for 5 min. The

supernatant liquid was further diluted to suitable volume

with water on the basis of the concentration of AA in fruits

given in the literature. 1 mL aliquot was analyzed as de-

106 J. Chin. Chem. Soc., Vol. 57, No. 1, 2010 Tiwari

scribed above.

Determination of AA in biological samples

Since the presence of AA has been reported in blood

and urine samples.14,21,22 The method was applied for its de-

termination in these samples. 5 mL each of blood and urine

samples were taken from pathology laboratory and 1 mL of

5% EDTA, 2 mL of 1% TCA (trichloroacetic acid) and 2

mL of 3% metaphosphoric acid were added to the analyte,

centrifuged, the supernatant was diluted to a suitable vol-

ume and 1 mL aliquot was analyzed as given in the proce-

dure.

RESULTS AND DISCUSSION

Absorption Spectra

The reaction of AA with potassium iodide-potassium

iodate mixture solution in acidic medium, liberated io-

dine.21 The liberated iodine selectively oxidizes the LMG

to MG dye (Scheme I). The green colour of the dye was de-

veloped in an acetate buffer (pH-4.0-4.8) on heating in a

water bath (~40 �C) for 5 min. A time period of 5 min was

required for complete colour development after dilution to

25 mL. The MG dye showed maximum absorbance at 620

nm, and the reagent blank had negligible absorbance at this

wavelength (Fig. 1).

Effect of the reagents concentration

Constant and maximum absorbance values were ob-

tained when 0.4 mL of potassium iodide-potassium iodate

mixture solution (5:1), 1 mL of 0.02-mol l-1 hydrochloric

acid solution, 1 mL of 0.05% LMG solution and 2 mL of 1

M sodium acetate were added in the described order to get

maximum sensitivity. The presence of excess potassium io-

dide-potassium iodate mixture had no effect on the oxida-

tion of LMG under given experimental condition.

Effect of pH

The formation and stability of MG dye depends on

pH of the medium. The effect of pH was studied by varying

the acid concentration with a constant acetate buffer vol-

ume and vice versa. It was found that maximum coloration

of MG dye developed in the pH range 4.0-4.8. A decrease

in absorbance values was observed below and above this

pH range. Hence, an optimum pH of 4.5 � 0.2 was main-

tained throughout the study by using acetate buffer.

Effect of time and temperature

It has been observed that the colour development was

rapid at higher temperature. Under optimum condition ~40

�C was most suitable. So that the reagent system required

heating in water bath for 5 at min ~40 �C an increase of

temp (> 40 �C) and the duration of heating (> 5 min) mark-

edly affect the sensitivity and reproducibility of the colour

system. It was also found that a time period of 10 min was

required for complete colour development after dilution to

25 mL. The formed dye was stable for several days.

Analytical data

The adherence to Beer’s law was studied by measur-

ing the absorbance values of solutions varying AA concen-

tration. A straight-line graph was obtained by plotting the

absorbance against the concentration of AA. Beer’s law

was obeyed in the concentration range of 0.8-8.0 �g AA per

Determination of Ascorbic Acid J. Chin. Chem. Soc., Vol. 57, No. 1, 2010 107

Fig. 1. Absorption spectra of MG dye.

25 mL of final solution. The apparent molar absorptivity,

and Sandell’s sensitivity of the method were found to be

2.98 � 105 l mol-1cm-1, 0.0042 �g cm-2, respectively. The

reproducibility of the method was checked by seven repli-

cate measurements, each containing 4.0 µg ascorbic acid

per 25 mL of final solution, standard deviation and relative

standard deviation were found to be � 0.0076 and � 1.6%,

respectively. The lower relative standard deviation value (�

1.6%) and the range of error at 95% confidence level in

terms of absorbance were � 0.0070, indicating good preci-

sion of the method.

Interference Studies

The validity of the method was assessed by investi-

gating the effect of various diverse ions and interfering spe-

cies in the analysis of AA. The tolerance limit of different

foreign species in a solution containing 4.0 µg per 25 mL of

ascorbic acid, causing an error of not more than � 2.0% in

the absorbance values are given Table 1. Species such as;

folic acid, ferrous fumarate, vitamin B1, B2, B6, B12, nico-

tinamide, and calcium pentothenate that are commonly

present in pharmaceutical preparation and number of for-

eign species such as; oxalic acid, citric acid, succinic acid,

tartaric acid, malic acid, lactic acid, glucose, sucrose, fruc-

tose, calcium chloride, sodium chloride that are known to

be present in fruit juices do not interfere with the proposed

method. Interference of metal ions such as; Fe3+, Al3+, Zn2+,

etc were prevented by the addition of 1 mL of 5% EDTA so-

lution. Most of the common ions and other major toxicants

did not interfere in the method under optimum conditions

employed.

Applications

The method has been applied satisfactorily to the de-

termination of AA in fruit juices, pharmaceuticals, and bio-

logical samples. To check the accuracy and reliability of

the method, vitamin-C, multi-vitamin and calcium tablets

of different brand were analyzed by this method. The re-

sults were in good agreement with the claimed value. Fur-

ther to ensure the accuracy and reliability, recovery tests

were performed by the standard addition of ascorbic acid to

the various environmental samples. The recovery values in

percent, obtained were quantitative and in good agreement

with reference method1,16,21 and this showed that the method

works satisfactorily (Tables 2, 3 and 4).

CONCLUSIONS

This article reports the use of leuco malachite green

for the first time as a new reagent for the spectrophotomet-

ric determination of AA. It offers a sensitivity, selectivity,

simplicity and cost-effectiveness of the method. The method

involves no extraction steps, thereby the use of organic sol-

vents, which are generally toxic in nature are avoided. The

stability of formed MG dye is an added advantage of the

method. The sensitivity in terms of molar absorptivity and

precision in terms of relative standard deviation of the pres-

ent method indicated it to be very reliable for the determi-

108 J. Chin. Chem. Soc., Vol. 57, No. 1, 2010 Tiwari

Table 1. Effect of foreign species (4.0 �g of AA per 25 mL)

Foreign Species

Tolerance

limit*

�g mL-1

Foreign Species

Tolerance

limit*

�g mL-1

Nicotinamide 3550 Na+, K+, Ba2+,

Ca2+

3400

Ferrous fumarate 3340 F-, Cl-, Br-, PO43- 2300

Citric acid, glucose,

sucrose, fructose

3000 SO42-, No2

- 2000

Acetate, tartaric acid,

malic acid, lactic acid,

Succinic acid

1500 Mg2+, Na+ 1000

Calcium pentothenate 1000 Zn2+, Fe3+, Al3+ 0400

Vitamin B1, B2 0950 Co2+, Cu2+

B6 0400

B12 0150

Folic acid 0070

* Tolerance limit is the amount of foreign species that causes an

error of � 2% in absorbance value.

Table 2. Results of determination of AA contents in pharma-

ceutical

Ascorbic acid mg per tablet*

Vitamin CClaimed

Value

Proposed

method

Reported

method1

Brand - A 100 099.90 (� 0.092) 099.12 (� 0.015)

Brand - B 500 499.94 (� 0.019) 499.34 (� 0.026)

Multi-vitamin preparation

Brand - A 150 149.96 (� 0.022) 149.89 (� 0.035)

Brand - B** 075 074.95 (� 0.012) 074.85 (� 0.019)

Brand - C*** 025 024.78 (� 0.052) 024.74 (� 0.015)

Calcium tablet 150 149.98 (� 0.022) 149.86 (� 0.021)

* Mean, � standard deviation of five replicates, ** content in mg

per capsule (containing ferrous Fumarate), *** content in mg per

capsule.

Determination of Ascorbic Acid J. Chin. Chem. Soc., Vol. 57, No. 1, 2010 109

Table 3. Determination of AA in fruit juices

Ascorbic acid originally

found*

Samples*Proposed

Method

(�g)

(A)

Reported

method16

(�g)

Ascorbic

acid added

(�g)

(B)

Total

Ascorbic acid

found by

proposed

method

(C)

Difference

(C-A)

Recovery**

%

(C-A)

(B)� 100

Orange 5.24 5.23 3.0 8.22 2.98 99.33

Lemons 5.83 5.85 3.0 8.81 2.98 99.33

Tomatoes 3.96 3.92 2.0 5.92 1.96 98.00

Grapefruit 2.78 2.78 2.0 4.76 1.98 99.00

* aliquot of sample- 1 mL, ** mean of five replicates

Table 4. Results of determination of AA in blood and urine

Ascorbic acid originally found

(�g)

Samples* Proposed

Method

(A)

Reported

Method21

Ascorbic acid

added (�g)

(B)

Total ascorbic

acid found by

proposed method

(�g)

(C)

Difference

(C-A)

Recovery**

%

(C-A)

(B)� 100

Blood 1.56 1.55 2.0 3.54 1.98 98.00

Urine 1.45 1.42 2.0 3.42 1.97 98.50

� aliquot of sample- 2 mL, ** mean of five replicate

Table 5. Comparison of present method with other spectrophotometric method

Methods/References � max nmBeer’s law range/

detection limitRemarks

Potassium chromate-

diphenylcarbazide1

548 5 �g mL-1 Method is indirect and less

sensitive.

2,4-dinitrophenyl

hydrazine13

524 8 �g mL-1 Only applicable to biological

materials, less sensitive.

2,6-dichlorophenol-

indophenol sodium15

520 1 �g mL-1 Subject to limitation, less

stability of dye, and serious

interferences of species that

reduce DCDIP.

Silver gelatin complex16 415 1-10 �g mL-1 Reagent is expensive and less

sensitive.

Tris, 1,10-phenonthroline

complex17

510 50-400 �g mL-1 Applied for determination to

wide range and less sensitive.

Fast red20 630 5-25 �g mL-1 Colour is stable for only 2 h

and less sensitive.

Leucocrystal violet21 590 0.01-0.1 �g mL-1 Though method is highly

sensitive but the reagent used

is costly and not easily

available.

Leuco malachite green

(Present Method)

620 0.032-0.32 �g mL-1 Simple, highly sensitive, cost

effective, higher stability of

colour, no need extraction in

to the organic phase and

applicable to wide range of

samples.

nation of AA in various samples. This method is good alter-

native to some reported costly instrumental method. The

results summarized in Tables 2, 3, 4 and 5 clearly showed

that the developed method worked satisfactorily.

ACKNOWLEDGEMENT

The author KKT is thankful to Prof. H. S. Kar Head,

Department of Chemistry & Biochemistry, Government

College of Science, Raipur and University Grant Commis-

sion, New Delhi for providing laboratory facilities and fi-

nancial assistance to carry out this experiment.

Received July 9, 2009.

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