"CHAPTER I"

Methodology For The Determination

Of Ascorbic Acid
 Introduction

In the determination of ascorbic acid, advantage is

taken of the chemical characteristic of the two dienol

groups on the carbon atoms 2 and 3. These groups have

two easily oxidisable hydrogen atoms which make possible

©f the application of considerable number of procedures

based on oxidation-reduction.

(i) Oxidation-Reduction Method :

Several reagents have been proposed for measuring

the oxidation of two loosely bonded hydrogen atoms of the

dienol groups, by various workers. The reagents used are

2,6-dichlorophenolindophenol (Tillmans, 1932), methylene

blue (Martini, 1934), iodine (Birch, 1953)* phosphomolybdate

(Bezssonoff, 1921), ferric chloride (Sullivan, 1953). Of

all the reagents tried, 2,6-dichlorophenolindophenol has

been found to be most suitable, as the dye indophenol has

an oxidation-reduction potential which is nearer to

oxidation-reduction potential of ascorbic acid. Moreover

the dye very specifically reacts with the extract containing

ascorbic acid at pH 3*5 • The other interfering substances

(phenolic group and sulphahydryl group) oannot react at this

low pH. This method is widely used and it only measures the

reduced ascorbic acid.

(ii) The Colorimetric Method Based on the Formation

of a 1H.nitrophenylhydrazine Derivative (Roe and

Kuether, 1943) t

In this method ascorbic acid is oxidised to dehydro-

ascorbic acid by certain suitable oxidants. The dehydro-

ascorbic acid so formed is then coupled with 2,4-dinitro-

phenylhydrazine. The solution is then treated with H^SO^

to produce a, red color which is measured photometrically.

This method measures the total ascorbic acid (ascorbic acid,

dehydroascorbie acid and diketogulonic acid).

Several reagents as oxidants have been suggested by

various investigators. .Among them those which have more

general applications are norit charcoal (Roe and Kuether,

1943) and bromine (Mills and Roe, 1947). In the present

study measurement of the pure ascorbic acid and the ascorbic

acid contents of different biological tissues (Liver, kidney

and intestine of rat) by using the above oxidants along with

the recovery experiments have been made in order to evaluate

the specificity of these reagents. In course of this

investigation, it has been observed that both the methods

have certain disadvantages.

Norit method s Although this method is generally

followed, but sometime reproducible results could not be

obtained owing to the nonavailability of standard charcoal

 /
44

in this laboratory. However, when the charcoal was heated

with 10 per cent HC1 (iron free) washed several times with

distilled water to remove iron and chloride and dried at

105°C, it gave consistent results. But it was rather a

long-drawn process and the charcoal sometimes lostes its

activity on keeping at room temperature for certain period

of time. Moreover, when the volume of tissue extract was

small, as in case of adrenal extract, norit charcoal was

not at all suitable, since the solution which remained

after titration with indophenol dye was not sufficient for

the estimation of total ascorbic acid by this method. It

is also known that activated charcoal adsorps oxidised,

ascorbic acid7hence the extraction of tissues must be

performed either in the presence of a mixture of metaphos-

phoric and acetic acid or trichloro acetic acid or oxalic

\
acid.

Bromine method t This method is also commonly used,

but it was not found satisfactory for reasons indicated below

i) After oxidation of ascorbic acid with bromine,

, complete removal of the excess bromine was not a

very quick process, especially, when several tissue

extractsware to be oxidised in this process.

ii) If the excess bromine is not properly passed off

by aeration, it in turn makes the solution turbid

 45

when reacted with dinitrophenylhydrazine. As a

result it records a higher ascorbic acid value.

iii) It is not a desirable reagent to use for this

purpose because of its physical and toxic

properties (Bolin and Book, 1947)•

iv) This method only measures the total ascorbic acid

content of the extract.

Bolin and Book (1947) proposed the use of 2,6-di-

chlorophenolindophenol as an oxidant for the estimation of

total ascorbic acid. It has been reported by Soe (1961)

that this reagent has no advantage as an oxidising agent

and the serious limitation of not providing for clarification

of the extract and removal of interfering substances. In

view of the certain disadvantage, regarding the estimation

of ascorbic acid by norit and bromine methods as described

earlier and since the question of clarification of the

extract of the aniital tissues does not arise, in the present

study the indophenol dye has been used as an oxidising agent

for the estimation of ascorbic acid. The method has, however,

been found to be most convenient because of its easy operatioc

and by this procedure one can measure both the reduced ascorbi

acid as well as total ascorbic acid simultaneously in the

same solution. In this operation the reduced ascorbic acid

is measured titrimetrically with the dye, in the same titrated

 46

solution the total ascorbic acid is determined by coupling

with 2,4-dinitrophenylhydrazine reagent. The difference

between the total ascorbic acid and reduced ascorbic acid

will give the values for dehydroascorbic acid and diketogu-

lonic acid, if any.

In the present investigation comparative studies

on the estimation of ascorbic acid by three different methods

have been made on the pure ascorbic acid solution as well as

in different tissues of rats. Experiments on the recovery

of added ascorbic acid from the tissues have also been

performed in order to ascertain the suitability of these

methods.

Experimental

Estimation of Ascorbic Acid by Titrimetric Method with

Indophenol Dye.

Principle of titrimetric method i It is based on

the reduction of the 2,6-dichiorophenolindophenol by an

ascorbic acid solution. In the absence of interfering

substances, the capacity of the extract of the sample to

reduce a standard solution ! of the dye which has been

standardised against pure'ascorbic acid solution is directly

propOrbioraaiirtdcthe.iascorbicitacid content.
 47

Reagents s /"~A11 chemicals used in the present and

subsequent experiments were analytical

reagent grade (BDH or E.Merck)_7

1. 5 per cent metaphosphoric acid (HPO^)

50 g of HPO^ was dissolved in approximately 900 ml

of glass distilled water and then made to 1 litre. HPO^

undergoes hydrolysis in 4 aqueous solution forming

S'

orthophosphoric acid, hence this solution was prepared weekly

and stored in the refrigerator.

2. 10 per cent metaphosphoric acid

3. Ascorbic acid standard solution

50 mg of ascorbic acid was taken in a 250 ml

volumetric flask and the volume made to the mark with

5 per cent metaphosphoric acid. 1 ml of this solution

contained 0.2 mg of ascorbic acid. It was freshly prepared

immediately before use for the standardization of

indophenol dye.

4. 2,6-Dichlorophenolindophenol

50 mg of sodium, 2,6-dichlorophenolindophenol was

taken in a beaker. To it 150 ml of glass distilled water

was added, warmed gently until dissolved and then added

42 mg of WaHCO^. The solution was cooled, filtered and

made to 200 ml with glass distilled water.

 48

5* 0.25 M sucrose solution

8.55 g of sucrose was dissolved in 100 ml of glass

distilled water.

Standardization of Indophenol Dye t

1 ml of the dye solution was taker, in a clean test

tube (5”x3/4m, pyrex)• The standard ascorbic acid solution

was allowed to fall dropwise from a 2 ml'graduated pipette

(100 division) to the dye solution with slight shaking of

the test tube. The addition of ascorbic acid solution was

continued until the solution turned colorless. The volume

of ascorbic acid solution required to decolorise was noted

and the strength of the dye was calculated..

Reaction Mechanism s

2,6-dichlorophenolindophenol is a dye that is •

blue in alkali and pink in acid. It'is reduced by

ascorbic acid to the colorless leuco:^orm, as shown by

the following reaction.

 h9

0=C
I
HO--C
II
HO-C
i
H-C
I
HO-C-H
ch2oh
2,6-dichlorophenolindophenol
(oxidised form,blue or pink)
Ascorbic Acid

CHgOH
2,6-dichlorophenolindophenol
(reduced form - colorless)
Dehydroascorbic Acid

Preparation of Tissue Homogenate i

Different tissues (liver, kidney, brain and

intestine) of rat were carefully weighed and homogenized

in ice cold isotonic sucrose solution (0.25 M) in the

ratio of 1:4 (w/v) in all glass Potter-Elvehjem

homogenizer for 2-3 minutes at 0-2°C.

Procedure for the Determination of Ascorbic Acid

In Tissue Homogenates s

Exactly 2 ml of tissue homogenate was taken in a

test tube. To it 2 ml of 10 per cent metaphosphoric acid

 • 50

was added to precipitate protein and filtered. The

filtrate was then titrated against a standard dye solution s

Titration s- 1 ml of the dilute standard indophenol

dye was taken in a test tube (5,,3:3/4,,, Pyrex). The filtrate

was then allowed to fall dropwise from a 2 ml graduated (lOO

divisions) pipette to the dye solution with slight shaking

of the tube, until the blue dye turned colorless. The value

of the filtrate required to decolorise 1 ml of standard dye

solution was noted and the amount of ascorbic acid present

in the filtrate was calculated.

Dinitrophenylhydrazine method (Colorimetric method of

Roe and Keuther, 1943) 5

Principle i-

In this procedure ascorbic acid is oxidised to

dehydroascorbie acid by mild oxidising agent. Dehydroascorbic

acid slowly undergoes to diketogulonic acid at low pH. "When

2,4-dinitrophenylhydrazine is added to dehydroascorbic acid

and diketogulonic acid, bis-2,4-dinitrophenylhydrazone

derivative is formed by coupling with 2 and 3 carbon atoms

of dehydroascorbic acid and diketogulonic acid as shown by

following reaction. When treated with 85 per cent HgSO^ the

bis-2,4-$initrophenylhydrazone of dehydroascorbic acid and

diketogulonic acid undergoes some molecular rearrangement

 51

and a highly stable reddish brown product is obtained which

absorbs light maximally at•500 to 550 nyu and is measured

in a photoelectric colorimeter (Klett Summerson).

Reaction Mechanism s

H H
0=C-.
0=i]
J, 0
0—0 ■
1 I + 0 2 H20
H- C—’
I
HO-C-H
™2°H 2,4-dinitrophenyl- NO 1
2 HO-C-H
hydrazine I
CH20H
Dehydroascorbie
Acid
Bis-2,4-dinitrophenylhydrazine
derivative

Norit Charcoal Method j

Reagents ; ^~A11 chemicals used in the present and

subsequent experiments were analytical

reagent grade (B.D.H. or B.Merck^/

1. 5 per cent metaphosphoric - 10 per cent acetic acid.

50 g of metaphosphoric acid sticks (HPO^) was

dissolved in about 800 ml of distilled water. To it 100 ml

of glacial acetic acid was added and the volume was made upto

1 litre with distilled water and it x\ras stored in refrigerato

 . 52

2. Ascorbic acid in metaphosphoric - acetic acid

solution

100 mg of" ascorbic acid was dissolved in 100 ml

of 5 Per cent metaph.osph.oric - 10 per cent acetic acid

solution. 5 ml of the standard solution was taken into

a 250 ml volumetric flask and was made to volume with

metaphosphoric-acetic acid solution mixture. 1 ml of

this solution contained 20 pi g ( 3 0.02 mg) of ascorbic

acid.

3. Ascorbic acid in sucrose solution

100 mg of ascorbic acid was dissolved in 100 ml

of sucrose solution (0.25 M). 10 ml of the solution was

taken into a 100 ml volumetric flask and was made to volume

with sucrose solution. 1 ml of this solution contained

100 pi g (= 0.1 mg) of ascorbic acid.

4. Acid washed Norit

50 g of commercial, activated charcoal was taken in

a flask (litre capacity). It was then boiled with 250 ml

of 10 per cent hydrochloric acid (HCl). The mixture was

filtered with suction. The norit cake was then transferred

to a large beaker and was thoroughly washed several times

with distilled water and filtered until the filtrate no

/

longer showed a test for chloride and for iron. It was then

dried in oven at 105°C and stored in a wide-moutherstoppered

bottle
 ,53

5. 2 per cent 2,k-dinitrophenylhydrazine-thiourea

solution

2 g of 2,4-dinitrophenylhydrazine was dissolved in

100 ml of* approximately 9N HgSO^. To it k g of thiourea was

added and was shaken until dissolved. It was then filtered

and stored in refrigerator. This reagent was filtered

occasionally before use.

6. 85 per cent H^SO^ .

7* 9N H2SG4

Procedure :

A. Preparation of Calibration Curve s To 30 ml of

diluted standard solution of ascorbic acid (20 yug/ml),

1.2 g of acid washed activated norit was added to oxidise

ascorbic acid. The mixture was shaken thoroughly and

filtered. The filtrate was taken into four pyrex test

tubes (20 ml capacity) by pipetting exactly 0.5» 1> 1*5

and 2 ml containing 10, 20, 30, 40 yaig of ascorbic acid

respectively. The volume of each tube was then made upto

h ml with the mixture of metaphosphoric-acetic acid solution.

To each of the four tubes 1 ml of dinitrophenylhydrazine-

thiourea reagent was added. The solutions of the tubes -

were mixed well and kept in an incubator, maintained at

37°C for exactly 3 hours along with a blank tube containing

 54

4 ml metaphospheric acid-acetic acid solu-ion and 1 ml

dinitrophenylhydrazine-thiourea reagent. After the

reaction was complete the tubes were removed and placed

in ice water bath. To each of the tube, 5 ml of 85 per

cent HgSO^ was added dropwise, very slowly. The tubes

were shaken well while in the ice water to obtain complete

mixing and then kept at room temperature i*28°C). The

color produced was then measured in photoelectric

colorimeter (Klett and Summerson) using green filter

(540 nyu ). The result is given in Figure (l).

Preparation of Tissue Homogenates t

A pooled sample of 20 per cent tissue homogenates

of 2-3 rats were prepared by the method as described before.

B. Estimation of ascorbic acid contents of different tUsuej

(liver, kidney, brain and intestine) of rats by

norit method s

10 ml of 20 per cent tissue homogenates were taken

in a test tube (pyrex, 80 ml capacity). The protein was
f

precipitated with 20 ml of 5 per cent metaphosphoric acid-

10 per cent acetic acid solution, and it was filtered. The

filtrate was treated with 0.8-1 g of norit charcoal and

filtered again. 2 ml of the filtrate was then taken for

the measurement of total ascorbic acid by the method as

described earlier. The results are given in Table (l).

 55

C. Recovery of ascorbic acid added to different

tissues (liver, kidney and intestine) of rats -

estimated by three different methods s

Ascorbic acid solution (prepared in sucrose solution)

in different amounts (40-100 pig ascorbic acid/ml of tissue

homogenate) was added to 20 ml of 20 per cent tissue

homogenates. The protein'was precipitated by the method

described earlier and filtered. A portion of the filtrate

was taken for the estimation of reduced ascorbic acid by

titrimetric method and the remaining solution was used for

the estimation of total ascorbic acid by three different

methods. The results are given in Table (2).

Bromine Method :

Reagents : 1. Bromine

Other reagents were the same as described

in the norit method.

Procedure ;

A. Preparation of calibration curve

30 ml of the standard ascorbic acid solution

(20 yug/ml) was taken in a pyrex beaker. To it 1 or 2

drops of bromine was added and stirred until the solution

was yellow. The solution was then decanted to pyrex test

 56

tube (80 ml) from the excess liquid bromime, and a current

of air, after bubbling through, a water trap was passed

through the solution until the color of the bromine was

completely removed. The solution in different amount was

taken and the total ascorbic acid contents were measured

by the procedure as described in norit me-chod. The result

is presented in Figure (l).

B. Estimation of ascorbic acid contents of different

tissues (liver, kidney, brain and intestine) of

rats by bromine method

10 ml of a 20 per cent tissue homogenates were taken

in a pyrex test tube (60 ml). The protein was precipitated

by tAe method as described before. The filtrate was

collected in a beaker (pyrex) and it was treated with

bromine. After removing the bromine as before, 2 ml of

bromine free filtrate was taken in a test tube and the

volume of the filtrate was made up to h ml with metaphosphorii

acid-acetic acid mixture. The total ascorbic acid content of

the filtrate was measured by the procedure described under

norit method. Results are presented in Table (i).

C. Recovery of ascorbic acid added tc different

tissues (liver, kidney and intestine) of1 rats

The procedure is exactly same as described in the

norit method, except that the oxidation of reduced ascorbic
 57

acid was made by bromine in place of norit charcoal. The

results are presented in Table (il).

Indophenoloxidation method

In this method ascorbic acid was oxidised by

indophenol dye before its coupling with 2,4-dinitrophenyl-

hydrazine.

Reagents t

1. 2,6-dichlorophenolindophenol

A standard indophenol dye solution which can

neutralize 180 ^mg of ascorbic acid per ml of dye was

prepared. A dilute solution of dye 10-20 yug ascorbic acid/

ml of dye was prepared. Other reagents are same as described

4..

in norit method.

Procedure i

A. Preparation of calibration curve

0.5> 1> 1.5 and 2 ml of the standard solutions

(20 yug/ml) were taken into four test tubes (pyrex).

Solution of indophenol dye (100 ^/ug of ascorbic aeid/ml)

was allowed to fall dropwise to each four tubes with light

shaking until the solution developed pink color. The dye

was added in slight excess to be sure of the complete

oxidation of ascorbic acid. The volume of each four tubes

 58

was then made upto 4 ml with metaphosphor^c-acetic acid

solution with a blank containing 4 ml of netaphosphoric

acid-acetic acid solution. The other procedures for the

determination of ascorbic acid were the same as described

earlier. The result is presented in Figure (l).

B. Estimation of ascorbic acid contents of different

tissues (liver, kidney, brain and intestine) of

rats by indophenol method

5 ml of a 20 per cent homogenate of tissues were

taken from a pooled sample and protein was precipitated

in the same method as described before and it was filtered.

The filtrate was titrated against a standard indophenol dye

as described in the titrimetric method. The titrated volume

of the filtrate was then made to 4 ml with metaphosphoric-

l

acetic acid solution and the other procedure was the same

as described in the norit method. The results are presented

in the Table (i).

C. Recovery of ascorbic acid added to different

tissues (liver, kidney and intestine) of rats

Ascorbic acid solution (prepared in sucrose solution)

was added in different amounts (40, 50, 60, 80, 100 yag

ascorbic acid/ml of tissue homogenate) to 5 ml of 20 per cent

of tissue homogenates. Protein was precipitated as before

 59

The reduced ascorbic acid content of the filtrate was

estimated by titrimetric method and total ascorbic acid

content of the titrated sample was determined by the method

described above. The results are presented in Table (il).

Results s

Estimation of ascorbic acid contents of different tissues

(liver, kidney and intestine) of rats by indophenol,

norit and bromine methods.

It will be seen from Table (l) that there is little

difference in the values of total ascorbic acid content in

the liver, kidney, brain and intestinal tissues of rats,

measured by three different techniques.

Recovery of ascorbic acid added to different tissues(liver,

kidney said intestine) of rats - estimated by three different

methods.

It is observed that 95-109 per cent of the vitamin

added to the tissue homogenates can be recovered, when

measured by indophenol, norit and bromine methods (Table II)

It is, however, noticed that in the case of bromine method

there is slightly higher per cent of recovery.

 ,6o

TABLE - I

1 WJ 9

brain and intestine' of rat measured by

three different methods.

j i Tissue ascorbic acid levels ( /ug/ml

No. of
obser-
I Tissue | tissue homogenate) '

vation
I Norit
j Charcoal |Bromine
’I Indophenol Method
J Method
| Method
i Reduced Total j Total Total
ascorbic jascorbie 1 ascorbic
I I ascorbic
acid acid ! acid I acid

1a Liver 59 60 59 66

1b II
53 52 52 58

1c If 49 50 49 55

2a Kidney 40 41 40 47

2b n
37 36 36 42
ii
2c 37 35 35 43

3a Brain 82 80 81 87
3b II
79. 80 78 84
it
3c 79 79 77 85

4a Intestine 64 62 63 69

4b II
59 61 58 65

4c II
70 65 68 74
 TABLE - II

Recovery of ascorbic acid added to different tissue (liver, kidney and intestine)homogenates
of rat estimated by three different methods.

A s c o rb ic A s c o r b i c A c id r e c o v e r e d ( y u g /m l t i s s u e h o m o g e n a te )
a c id N o rit c h a rc o a l
N o. of added I n d o p h e n o l M e th o d M e th o d B r o m in e M e th o d
o b s e r­ T is s u e ( /u g /m l
v a tio n T o ta l P e r c e n t ' a Ts coot ar bl i c P e r c e n t
' t i s s u e Ra se cd ou cr be di c a Ts coot ar bl i c P e r c e n t
re c o v e ry a s c o r b i c re c o v e ry
hom oge­ a c id a c id a c id a c id re c o v e ry
i
n a te )
i
i
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61
H E A D IN G
KLETT

0 10 20 30 40
ASCORBIC ACID IN jJi g

FIG. 1

STANDARD CURVES OF ASCORBIC ACID OBSERVED BY

THREE DIFFERENT METHODS.
 62

Discussion i

A comparative study on the estimation of ascorbic

acid in pure solution as well as in the different tissue

homogenates of rats by using three different techniques,

showed no significant difference in their results. Further,

from the recovery experiment it was found that 95-99 per

cent added ascorbic acid could be recovered from tissue

homogenates. The estimation of ascorbic acid by bromine

£-tf v1
method, however, gave slightly higher results particularly

in the tissues, which might have occured due to incomplete

removal of bromine from the samples. Hsien-Sheng P’eng

(1958) reported a similar recovery (95-99 per cent) of

added ascorbic acid, titrated with indophenol dye. The

modified method of estimation of ascorbic acid (indophenol

method) as suggested in this present investigation was

found to be more convenient and suitable incomparison to

other two methods. Since in this method it was possible

to determine both reduced and total ascorbic acid in a

small tissue extract.

Summary s

A simple method of estimation of reduced and total

ascorbic acid by using indophenol dye has been developed.

The method was compared with other two standard methods

(norit charcoal and bromine) including recovery experiments.
 63

The method was found to be most suitable, because

of its quick and easy operation. It has another advantage

that both the reduced and total ascorbic acid could be

measured in a very small amount of tissue extract.

5?