ASCORBIC ACID

Ibrahim A . Al-Mesh1 and Mahmoud M . A. Hassan

1. Description 46
1.1 Nomenclature 46
1.2 Formulae 46
1.3 Molecular Weight 47
I .4 Elemental Composition 47
1.5 Appearance, Color, Odor, and Taste 47
2. Physical Properties 47
2.1 Crystal Properties 47
2.2 Solubility 48
2.3 Specific Rotation 48
2.4 Dissociation Constant 49
2.5 Identification 49
2.6 Spectral Properties 51
3. Preparation 61
3.1 Isolation 61
3.2 Synthesis 63
4. Biosynthesis of Ascorbic Acid 63
5. Metabolism 66
6. Daily Requirement 66
7. Mode of Action 66
8. Vitamin Deficiency 67
9. Methods of Analysis 67
9.1 Titrimetric Methods 67
9.2 Spectrophotometric Methods 70
9.3 Turbidimetric Method 73
9.4 Chromatographic Methods 73
9.5 Enzymatic Method 75
9.6 Polarographic Method 15
9.7 Chronometric Method 75
10. References 76

Copyrighl 0 1982 by The Amenan

Analytical Profiles of Drug Substances phrrm.ceuUcd Awduion
Volume II 45
ISBN 0-12-260811-9
46 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

1, Description

1.1. Nomenclature

1.1.1 Chemical Names

a) L-Ascorbic a c i d
b) L-Xyloascorbic a c i d
c) 3-0x0-L-gluofuranolactone (enol form).
d) L-3-Ketothreohexuronic a c i d l a c t o n e

1.1.2 Generic Names

Vitamin C ; A s c o r b i c a c i d

1.1.3 Trade Names

Adenex; A l l e r c o r b ; A n t i s c o r b u t i c Vitamin;
Ascorbicap; Ascorbajen; A s c o r i l ; A sc o ri n ;
A s c o r t e a l ; A s c o r v i t ; Cantan; C a n t a x i n ; Cata-
v i n C ; Ce b i c u r e ; Cebid; Cebione; Cecon;
Cegiol an ; Cell i n ; Cenetone; Cereon; Cergona;
Ce s c o r b a t ; Cetamid; Cetan; Cetemican; Ceva-
l i n ; Ce v a t i n e ; Cevex; Cevimin; Cevi-Bid;
Ce-Vi-Sol; Cevitamin; C e v i t a n ; Cimin; C e v i t a -
mic Acid; C e v i t e x ; Ciamin; C i p c a ; C o l a s c o r ;
Concemin; C-vimin; Davitamon C ; E r i v i t C ;
Hybrin; L a r o s c o r b i n e ; Lemascorb; Megascorb;
P l a n a v i t C ; Pr o s c o r b i n C ; Redoxon; Ribena;
Sc o r b a c i d ; Scorbu-C; T e s t a s c o r b i c ; V i c e l a t ;
V i t a c e ; V i t a c i mi n ; V i t a c i n ; V i t a s c o r b o l ;
Vitix.

1.2 Formulae

1.2.1 Empirical

‘gH8’6

HR=o
1.2.2 Structural CtI*OtI
I
HOCH

HO OH
ASCORBIC ACID 41

1.2.3 CAS No.

(50-81-7)

1.2.4 (Wiswesser Line Notation

TOSV EHJ CQ DQ EYQ IQL (1)

1.2.5 Stereochemistry

The nature o f the ring system in ascorbic

acid was determined by a study of the methy-
lated derivatives of the acid. By this means
complete confirmation was obtained of the
accuracy o f the views advanced, concerning
the stereochemical configuration of the
molecule and the nature of the reactive
enolic groups(2). The furanose structure for
ascorbic acid shown above (1.2.2) was put
forward by Lerbert el a1 ( 2 ) on the basis
o f its chemical behaviour as well as its
oxidation products.

1.3 Molecular Weight

176.12

1.4 Elemental Composition

C, 40.91%; H, 4.58%; 0 , 54.51%.

1.5 Qpearance, Color, Odor and Taste

White or slightly yellow crystals or powder. Odor-

less or almost odorless; pleasant sharp acidic
taste(3).
2. Physical Properties

2.1 Crystal Properties

Usually plates, sometimes needles, monoclinic

system (4).

2.1.1 X-ray Diffraction

The available data (2) of the X-ray, reveals

that the total of 12 carbon and oxygen atoms
48 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

all but one can be accomodated in one plane

without appreciable valency strain whilst
the remaining carbon (C5) lies less than 1A"
above the plane as shown in the following
model :

2.1.2 Melting Range

Ascorbic acid melts at 190-192" with decom-
position ( 4 ) .

2.2 So lubi1ity

Ascorbic acid is soluble at 20", in 3.5 parts of

water and 25 parts of alcohol (95 per cent); 50
parts of absolute alcohol, 100 ml of glycerol, 20 ml
of propylene glycol. Solubility in hot water 40.0%
at 40°, 80% at 100". Insoluble in ether, chloroform,
benzene and light petroleum (boiling range 40-60").
2.3 Specific Rotation

[a] is+ 20.5" to 21.5" (C = 1, water)

[a] i3+ 48 (C = 1, methanol) (4).

l9 + 24 (water) (2).
['I 5780
l8 + 116 (sodium salt in neutral solution) ( 4 )
5780
l8
+ 130 (N/20 NaoH) ( 2 ) .
5780
(2).
5780 + 149 (N/l NaoH)
l8

I'[ l8
5780
+ 155 (N/2 NaoH) (2)

l8 + 161 (2N NaoH) (2)

5780
ASCORBIC ACID 49

2.4 Dissociation Constant

Ascorbic acid is a moderately strong organic acid,

two ionization constants:

pK1 4 . 1 7 and pK 11.57. pH = 3 (5mg/ml), pH = 2

2
(50 mg/ml) (4).
2.5 Identification

i) Solution of ascorbic acid decolorises, 2,6-

dicholorophenol-indophenol solution (5).

ii) Solution and ascorbic acid reduces silver

nitrate solution immediately in the cold
producing a black precipitate (5).

iii) Dissolve 0.1 g of ascorbic acid in sufficient

w a t e r to produce 100 ml and dilute 1 ml to
100 ml with 0.01M h y d r o c h l o r i c a c i d . The
light absorption of the resulting solution
exhibits a maximum only at 244 nm; A (1 per
cent, 1 cm) at 244 nm, about 560 (6).

iv) To 2 ml of a 5 per cent w/v solution add 0.5

g of s o d i u m h y d r o g e n c a r b o n a t e ; carbon
dioxide is evolved ( 6 ) .

v) To 1 ml of a 5 per cent w/v solution add

about 0.2 ml of ZM n i t r i c a c i d and 0.2 ml of
0.1M silver n i t r a t e ; a grey precipitate is
produced (6).

vi) To 5 ml of a 1 per cent w/v solution add

0.05 ml of a freshly-prepared 5 per cent w/v
solution of s o d i u m n i t r o p r u s s i d e and 2 ml of
2M s o d i u m h y d r o x i d e followed by 0.6 to 0.7
ml of h y d r o c h l o r i c a c i d , added dropwise with
stirring; the yellow color turns blue (6).

vii) Specific optical rotation, in a 10 per cent

w/v solution, +20.5' to +21.5O (6).

viii) A solution (1 in 50) reduces alkaline cupric

tartrate TS slowly at room temperature but
more readily upon heating ( 7 ) .
50 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

ix) To 2 ml of a solution (1 in 50) add 4 drops

of methylene blue TS, and warm to 40": the
deep blue color becomes appreciably lighter
or is completely discharged within 3 minutes
(7).

XI Dissolve 15 mg in 15 ml of a solution of
trichloroacetic acid (1 in 201, add about
200 mg of activated charcoal, shake the
mixture vigorously for 1 minute, and filter
through a small fluted filter, returning the
filtrate, if necessary, until clear. To 5 ml
of the filtrate add 1 drop of pyrrole, and
agitate gently until dissolved, then heat
in a bath at 50": a blue color develops ( 7 ) .

xi) For the examination of vegetable infusions

for the presence of vitamin C, the ascending
- descending paper-chromatographic method of
Block was used on the dinitrosazones. Many
combinations of solvent were used, but a
mixture of xylene and nitrobenzene (95:5)
were the most satisfactory. Of these, the
latter solvent furnished compact spots of
such definition that treatment with alcoho-
lic potash to reveal them was unnecessary
(8)

xii) Amounts of vitamin C down to 3 pg can be

detected as dark areas on the layer exposed
to short-wave UV light; brief heating to
120°C renders it fluorescent in radiation of
365 nm ( 9 ) .

xiii) The customary identification o f free ascor-

bic acid depends on its strong reducing pro-
perties and any of the reactions known from
paper chromatography may be utilized. The
limit of detection with indophenol reagent
(blue) is around 0.1 pg dipyridly-iron
(red) and molybdophosphoric acid (blue) are
almost as sensitive; after brief heating,
derivatives and decomposition products
yield the colors also. Amounts of 3-5 pg
can be visualized with iodoplatinate reagent
(yellow) and with alkaline silver nitrate
reagent (9).
ASCORBIC ACID 51

2.6 Spectral Properties

2.6.1 Ultraviolet Spectrum

The UV spectrum of ascorbic acid (0.002%) in

aqueous, acidic methanolic, ethanolic and
alkaline solution was scanned from 200 to 400
nm using Varian Carry 119 Spectrophotometer
(Fig. I ) . The UV maxima are as follows:

'max (nm)

Aqueous solution 263

Acidic solution 243
Methanol 244
Ethano 1 245

Other reported data (2) are as foJlows:

'max (nm>

Aqueous solution 260-265

Acidic solution (pH3) 245
Ethano 1 245
Methanol 263
Sodium salt in aqueous solution 265

2.6.2 Infrared Spectrum

The IR spectrum of ascorb,icacid as KBr-disc

was recorded on a Perkin-Elmer 580B FT-
spectrometer (Fig. 2 ) . The structural assign-
ments have been correlated with the following
band frequencies (Table 1).

Table - 1: IR Characteristics of Ascorbic

Acid,
Frequency Cm-I Assignment
3510
3405 OH
3306
1755 c=o
1670
1110
c-0-c
1025
52 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

. . .
200 210 225 230 2 4 250 260 270 284 2YO 300 310 2 0 3% 3u) 350 3m 380 390

Fig. 1. UV Spectrum of Ascorbic Acid. Ascorbic Acid

i n Water; --- Ascorbic Acid i n Ethanol; +scorbic Acid i n
-
Methanol; ....
Ascorbic Acid i n Acid Solution.
 1
4w mnmlmbr(tlDm am fWe mr f4a c w I a o c o o 4 m 2 I

Fig. 2. IR Spectrum of A s c o r b i c A c i d as KBr d i s c .

54 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

Other characteristic absorption bands are:

3208, 1500, 1390, 1372, 1320, 1275, 1250,

1222, 1200, 1140, 1075, 1068, 1045, 1025,
990, 870, 820, 755, 720, 680.

2.6.3 Nuclear Magnetic Resonance Spectra

2.6.3.1 Proton Spectra

The PMR spectra of ascorbic acid in

dueterium oxide,in pyridine and in
pyridine D5 were recorded on a
Varian-T-60-A, 60 MHz spectrometer
using sodium-2,2-dimethyl-2-sila-
pentane-5-sulphonate and tetramethyl-
silane as reference standard respec-
tively (Fig.3, Fig. 4 and Fig. 5).
The following structural assignments
have been made (Table 2):

Table - 2: PMR Characteristics o f Ascorbic

Acid.

Chemical Shift (ppm)

D20 Pyridine Pyridine D5 Assignment

4.87(d) 5.43(d) 5.36 (d) 5-H

'
4.10(m) 4.68(m) 4.33 (m) 6-H
3.77(s) 4.40(s) 4.36 (s)
3.68(d) 4.30(s) 4.23 (d) 7-CH2

s=singlet, d=doublet, m=multiplet.

 400

Fig. 3. PMR Spectrum of A s c o r b i c A c i d in D20.

I
L
1
0
z
L
I
0
m
0
0
;I
0
m
56
m
4

Fig. 5. PMR Spectrum of Ascorbic Acid i n Pyridine D5.

58 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

PMR data in D20 and in a mixture of

DMSO D6 and CDC13 were also reported
(10,11,12).
13
2.6.3.2 C-NMR

The 13C-NMR completely decoupled and

off-resonance spectra are shown in
Fig. 6 and Fig. 7 respectively. Both
were recorded over 5000 Hz range in
dimethylsulfoxide on Jeol FX-100, 100
MHz spectrometer. Using 10 mm sample
tube and tetramethylsilane as refe-
rence standard, at ambient tempera-
ture. The carbon chemical shifts are
assigned on the basis of the additi-
vity principles and the proton-
coupled spectrum (Table 3).

11
I
HO - c 6-
I

Table - 3: Carbon Chemical Shifts of Ascorbic

Acid.

Carbon No. Chemical Shift ppm

c-1 170.31(s)
c-2 117.93 ( s )
c-3 152.62 (s)
c-4 74.56 (d)
c-5 68.42 (d)
C -6 61.93(t)

s=singlet; d=doublet; t=triplet

13C-NMR data in Water have been also

reported (1 3) ,
 Fig. 6. 13C-NMR Spectrum of Ascorbic Acid (Completely de-
coupled).
 ~~

Fig. 7 . 13C-NMR Spectrum of Ascorbic Acid (off-Resonance) .

 61
ASCORBIC ACID

2.6.4 Mass Spectrum

The mass spectrum o f ascorbic acid obtained
by electron impact ionization, was recorded
on a Ribermag R-10-10 mas spectrometer equip-
ped with direct inlet probe. The spectrum
(Fig. 8) shows a molecular ion peak M+ at m/e
176 with a relative intensity o f 5.9%. The
most prominent fragments and their relative
intensities are shown in Table 4:

Table - 4: Prominent Mass Fragments of Ascorbic

Acid.

M/e Relative Intensity % Fragment

177 1.0 M + l
176 5.9 M +
1

116 100.0

85 36.0

71

70
24.7

23.1
HF
H
H o g

61 29.7
a'
HO-
'i'
C
PH
- 0
C
I I
H H
3. Preparation

3.1 Isolation

Many methods were reported €or the isolation o f ascor

bic acid from plants. However, the most popular is
by using freshly prepared solution of 5-6% methaphos-
phoric acid (14). This solution is a good extractant
as well as stabilizing agent f o r a limited period by
complexing metal ions and minimizing the rate of oxi-
dation. It has also been claimed that ascorbic acid
can be stablized by diluted perchloric acid solution
ASCORBIC ACID 63

o r 2,3-dimercapto-l-propanol (15). An alternative

method of extracting ascorbic acid from foods is by
forming a slurry of the frozen material with absolute
ethanol has been found to be as effective as extrac-
tion with metabphosphoric acid (16).Also a mixture
of 8% acetic acid and 0.5% oxalic acid was used (17)
3.2 Synthesis

L-ascorbic acid is conventionally synthesized (18,19)

by hydrogenating D-glucose to D-sorbitol. The latter
is made to yields L-sorbitol by oxidation with Aceto-
bacter suboxydan, this followed by introducing carbo-
xyl group at C 1 while the L-sorbose is in the form of
its diacetone derivative. The resulting diacetone-2-
keto-L-gluconic acid is then heated with hydrochloric
acid t o give ascorbic acid (Scheme 1 , p . 11).
Alternative route from sorbose by oxidation with
nitrogen peroxide.
Another method for synthesizing L-ascorbic acid was
reported ( 2 0 ) , involving a one-step oxidation of 1,2-
0-isopropyhene-a-D-glucofuranose to 1,2-0-isopropyli-
dene-a-D-xylo-hexofuranurono-6,3-Lactone-S-ulose and
acid treatment of the later followed by reduction.
4. Biosynthesis of Ascorbic Acid

In both plants and animals ascorbic acid is formed from D-

glucuronic acid. UDP-glucuronic acid is first converted to
D-glucuronic acid lactone via D-glucuronic acid-l-phos-
phate. This compound is then reduced at carbon atom 1 t o
form L-gulonic acid. (Since in the numbering of the carbon
atoms of carbohydrates the most highly oxidized carbon
atom is given the lowest possible number, the original
carbon atom 6 of glucuronic acid becomes carbon atom 1 of
gulonic acid). After the conversion of the gulonic acid
to the corresponding y-lactone, the hydroxyl group at
carbon atom 2 is oxidized to a keto group. The 2-keto-L-
gulonic acid lactone formed is subsequently converted t o
L-ascrobic acid by enolization (21). Direct conversion of
D-glucuronic acid to L-gulonic acid by isomerization at
carbon atom 5 has not yet been conclusively established
(scheme 2,~.12).
64 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

CH20H
CH20H CH20H
I I I
HO - C -
I
H IIO - C - H co
HO - C - H I I
HO- C- H HO- C--B
I H? I Acetobactv I
H -C-O€i B- C- OH H- C- OH
I cat. I I
HO -c - B HO - C - I-;
suboxydans
HO - c - H
I I I
CHO
Ct120H CH20€i
D-glucose
G-sorbitol L-sorbose
COOH
I
co
I
HO - c-
I
acetone + (1)*<Mn04 ~

ki - C - OH

I
H+ ( 2 ) H 2 0 (H+) I
H - -E
9:CMe2 HO C
I
CH OII
2,3,4,6-diacetone sorbose 2
2-keto-L-gulonic acid

(CH20H

bo
C -OH

HO-
II
C
11 I
eno-
lize
I lactoniz
H - C-OH
I H
HO- C-H OH O€i
HO-C-H
I I
I
CH20H
CH20H
L-ascorbic acid
(Vitamin C)
Scheme 1: Synthesis of ascorbic acid.
ASCORBIC ACID 65

-
0
II
..
6
COOH COOH C

Q"Dp-
HO
OH
Ho@o-p
OH
,@"20H
OH
UDP-D-Glycuronic D-Glucuronic acid- D-Glucuronic
acid 1-P acid lactone

CH20H CH20H
L-Gulonic acid L-Gulonic acid
lactone

HO-C-H
I I
CH20H CH20H

2-Keto-L-gulonic L-Ascorbic
acid lactone. acid.

Scheme 2: The biosynthesis of L-ascorbic acid.

66 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

5. Metabolism
Ascorbic acid is readily absorbed and metabolised. However,
after oral administration of large quantities, only small
amounts are excreted in the urine while there is a steady
rise in the level of ascorbic acid in the plasma. If the
oral ingestion is continued for a sufficient period, the
plasma concentration rises to a maximum, after which a
rapid urinary excretion of a large part of the ingested
ascorbic acid occurs (22). Ascorbic acid (ASA) and dehydro-
ascorbic acid (DAsA) are metabolized by humans, and the
levels of AsA in blood were maximum at the 4th hr after
AsA administration but at 2nd hr after DAsA administration.
The amount of AsA and DAsA excreted in urine in 6 hr was
respectively. 60 and 70% of the amount excreted in 24 hr
after administration, About 30 and 40% of administered
dose were excreted by men and women respectively in 24 hr.
Of the vitamin excreted after administration of either
AsA or DAsA, about 90 and 10% were in the form of AsA and
DAsA, respectively (23).
The amino acids phenylalanine and tyrosine are not meta-
bolized completely in vitamin C-deficient individuals,
Under these conditions they are metabolized only partly
and are excreted in the urine as homogentisic, p-hydroxy-
phenylpyruvic and p-hydroxyphenyllactic acids. It appears
that vitamin C plays the role of a coenzyme in the meta-
bolism of tyrosine through its deaminated product, because
scorbutic liver slices cannot metabolize this amino acid
in the absence of this vitamin. Vitamin C in adequate
amounts delays the oxidation of epinephrine by the body
(24 1.
6. Daily Requirement!
Ascorbic acid is needed in daily quantities of about 70 mg
to sustain full stamina, and is an essential nutrient for
human beings. In the case of insufficiency the symptoms
of scurvy appears (21).

7. Mode o f Action
We have as yet no complete understanding o f the mode of
action of ascorbic acid. It is, however, known that this
substance is involved in certain hydroxylation reactions
which are catalysed by mixed function oxygenases in the
reduction o f folic acid to tetrahydrofolic acid as well
as well in the regulation of the redox equilibrium between
Fez+ and Fe3+ and Cu" and C U ~ +(21).
ASCORBIC ACID 61

8. Vitamin Defficiency

Patients placed on a diet deficient in vitamin C

exhibited the following: 1) 10 days, plasma fell to
a low level; 2) 30 days plasma level was zero; 3) 13
weeks, first clinical evidence of scurvy; 4) 132 days,
hyperkeratotic papules developed; 5) 141 days, wounds
failed to heal and 6) 162 days, perifollicular hemor-
rhages of scurvy developed; ascorbic acid value of
white cell platelets fell to zero. Loss of weight
occurred, accompanied by lowered blood pressure (24).

9. Methods of Analysis

9.1 Titrimetric Methods

British Pharmacopoeia 1973

The B.P. 1975 has described the assay

as follows:

Weigh and powder 20 tablets. Dissolve a

quantity of the powder equivalent to 0.15 g
of ascorbic acid as completely as possible in
a mixture of 30 ml of water and 20 ml of
d i l u t e sulphuric acid and titrate with 0.lN
anunonium c e r i c sulphate, using f e r r o i n sulphate
solution as indicator. Each ml of 0 . 1 1 m o -
niwn c e ri c sulphate is equivalent to 0.008806 g
of C6H806.

British Pharmacopoeia 1980

The B.P. 1980 has described the assay as

follows :

Dissolve 0.2 g in a mixture o f 80 ml of freshly

boiled and cooled water and 10 ml of M sulfuric
acid. Titrate with 0 . 0 5 M iodine VS using 1 ml
of starch solution as indicator until a persis-
tent blue color is obtained. Each ml of 0.05M
iodine VS is equivalent to 0.00881 g of C H 0
6 8 6'

United States Pharmacopoeia 1980

a) Ascorbic Acid: Dissolve about 400 mg of

ascorbic acid, accurately weighed, in a
68 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

mixture of 100 ml of carbon dioxide-free

water and 25 ml of 2N s u l f u r i c acid. Titrate
the solution at once with 0.1 N iodine VS,
adding 3 ml o f starch TS as the end-point is
approached. Each ml of 0.1 N iodine is
equivalent to 8.806 mg of C6H806.

b) Ascorbic acid injections: Transfer to a 100-ml

volumetric flask an accurately measured volume
of ascorbic acid injection, equivalent to about
50 mg of ascorbic acid and previously diluted
with water if necessary. Add 20 ml of metaphos-
phoric-acetic acids TS, dilute with water to
volume, and mix. Accurately measure a volume
of the dilution, equivalent to about 2 mg of
ascorbic acid, into a 50-ml conical flask, add
5 ml of metaphosphoric-acetic acids TS, and
titrate with standard d i c h l o r o p h e n o l - i n d o p h e n o l
solution until a rose-pink color persists f o r
at least 5 seconds. Correct for the volume of
the dichlorophenol-indophenol solution consumed
by a mixture of 5.5 ml of metaphosphoric-
acetic acids TS and 15 ml of water. From the
ascorbic acid equivalent o f the standard
d i c h l o r o p h e n o l - i n d o p h e n o l solution calculate
the ascorbic acid content in each ml o f the
injection.

Various other titrimetric methods of assay

of vitamin C in vegetable tissues, whether as
ascorbic acid or as total vitamin C (ascorbic
plus dehydroascorbic acids), were studied and
compared. For the extraction of the vitamin,
an aqueous mixture of 8 per cent, acetic and
0.5 per cent oxalic acids was used instead of
metaphosphoric acid. Hot and cold extractions
gave practically the same results, except with
hard, dry tissues, which required hot extraction.
To effect removal of colloidal matter, which
interferes with filtration and clarification
of the extract, it is considered advisable to
add 25 to 50 per cent o f ethanol. In the
presence of ethanol causes an error o f at least
2 to 12 per cent. To establish (visually) the
titration end-point in the indophenol method,
it is recommended that a standard having the
same composition as the sample under titration
ASCORBIC ACID 69

b u t p r e v i o u s l y o x i d i s e d w i t h i o d i n e and
mercuric a c e t a t e be employed. T h i s g e n e r a l l y
e n a b l e s 93 t o 100 p e r c e n t of v i t a m i n C e x p e r i -
m e n t a l l y added t o be determined. I n t h e method
of d e t e r m i n a t i o n with methlene b l u e , t h e r a t i o
o f methylene b l u e used t o v i t a m i n C c o n t e n t
d e c r e a s e s as t h e l a t t e r v a l u e i n c r e a s e s . T h i s
d e c r e a s e of t h e r a t i o i s small; t o o b t a i n s a t i s -
f a c t o r y r e s u l t s , t h e a l i q u o t t i t r a t e d should
n o t c o n t a i n >0.04 mg o f v i t a m i n C . V i s u a l
d e t e r m i n a t i o n o f t h e end-point w i t h t h e a i d
o f a s t a n d a r d i s a g a i n recommended. The i o d i -
m e t r i c method, with potassium i o d a t e was a l s o
s t u d i e d . The c o n c l u s i o n s drawn a r e : 1) t h a t
t h e indophenol method i s s u f f i c i e n t l y a c c u r a t e ,
i s t h e most simple method and h a s t h e w i d e s t
s p h e r e of a p p l i c a t i o n ; 2) t h a t t h e methylene b l u e
method g i v e s r e s u l t s 3 t o 10 p e r c e n t lower
t h a n t h e indophenol method and 3) t h a t t h e
i o d i m e t r i c method g i v e s r ? s u l t s 20 t o 40 p e r
cmt higher (17).

Reduction i s e f f e c t e d by adding M sodium

s u l p h i d e s o l u t i o n a c i d i f i e d with HC1, and
removing t h e e x c e s s o f s u l p h i d e w i t h M
e t h a n o l i c mercuric c h l o r i d e s o l u t i o n . Reduc-
t i o n i s complete i n 10 t o 15 min. C l e a r
f i l t r a t e s a r e r e a d i l y o b t a i n a b l e €or t i t r a t i o n
w i t h dichlorophenol-indophenol ( 2 5 ) .

Simple methods o f d e t e r m i n i n g a s c o r b i c
a c i d , t h i a m i n e and n i c o t i n i c a c i d a r e
applied t o mixtures containing t h e s e
vitamins with various pharmaceutical
p r e p a r a t i o n s . Ascorbic a c i d can b e
determined i o d i m e t r i c a l l y i n t h e p r e s e n c e
o f calcium l a c t a t e , p h y t i n g l u c o s e calcium
g l y c e r o p h o s p h a t e , c a f f e i n e sodium b e n z o a t e ,
n i c o t i n i c a c i d , amidopyrine o r t h i a m i n e .
For m i x t u r e c o n t a i n i n g a s c o r b i c a c i d and
nicotinic acid, the ascorbic acid i s deter-
mined i o d i m e t r i c a l l y and t h e t o t a l a c i d i s
70 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

then titrated against 0.1 N solution of

hydrochloric acid with phenol red as indi-
cator. Thiamine i s determined in the
presence of ascorbic acid by a modifi-
cation of the U.S.S.R. Pharmacopoeia
VIII argentimetric method ( 2 6 ) .
The platinum - tungsten bimetallic elec-
trode system is applicable to the
quantitative iodimetric determination
of ascorbic acid according to the B.P.
1953. The equivalence point is given
by the very sharp break in the titration
curve. The error, = f 0.0002 on samples
of 0.05 to 0.15 g is much less than for
the usual volumetric procedure (27).
Other Sitrimetric method for the deter-
mination of ascorbic acid is by involv-
ing two stage oxidation by potasium
iodate ( 2 8 ) .

9.2 Spectrophotometric Method

9.2.1 Colorimetric
An assay method described for ascorbic acid
involves the reaction with diazotised 4-
methoxy-2-nitroaniline in acid medium, and
subsequent development of a blue color in
alkaline solution. This color, with a maximum
absorbancy at 570 nm, i s compared with stan-
dards in suitable photo-electric colorimeter.
It can be carried out directly, e.g., in the
presence of dehydro-ascorbic acid and all
other vitamins. Its sensitivity permits the
determination of quantities down to 0.5 mg
with a low limit of 10 pg per ml, when a 50-
ml sample aliquot is used (29a).
9.2.2 Ultraviolet
In aqueous solution ascorbic acid is charac-
terised by a single very intense band with
its head at 260-265.nm. The molecular extinc-
tion coefficient is approximately 7000 for
solutions containing about 2 mg/100 cc. In
stronger solutions (ca.50 mg per 100 c.c.)
wide deviations from Beer's law are encoun-
ASCORBIC ACID 71

tered. But for concentrations ranging between

0.5 and 2.5 mg per 100 C.C. Beer's law holds
with sufficient exactitude to permit of the
use of spectrophotometric measurements for
quantitative estimations of concentrations.
The intensity of the band diminishes rapidly,
falling to half value in a few hours (decom-
position of ascorbic acid by oxidation (2).
Other color reactions have been proposed as a
basis for measuring ascorbic acid: reaction
with diazotised p-aminobenzoic acid to produce
a pink color and the interaction of ascorbic
acid with dimethoxydiquinone to form a reddish
-violet color which is measured spectrophoto-
metrically at 510 nm (29b).
Ascorbic acid, cystine, thioglycollic acid,
a-mercaptopropionic acid and sodium mercapto-
butane sulphonate are determined by the fluore-
scence produce by the reduction of sodium 1 : Z -
naphthaquinone-4-sulphonate (Folin's reagent)
in U.V. The use of p-chloromercuribenzoic
acid in the determination of ascorbic acid
with 2,6-dichlorophenol-indophenol below pH3-5
is recommended because of the extent of spon-
taneous fading of indophenol that occurs. p -
Chloromercuribenzoic acid does not affect the
reaction between indophenol and ascorbic acid,
but it inhibits almost entirely the decolori-
sation of indophenol by various interfering
substances (31).
9.2.3 Spectrofluorimetric

The reaction of dehydroascorbic acid with o-

phenylenediamine to give a fluorescent quinox-
aline was used as the basis of an assay to
determine y quantities of ascorbic and dehydro-
ascorbic acids, The development of the fluore-
scent derivative of the vitamin is prevented
by forming a boric acid-dehydroascorbic acid
complex prior to addition of the diamine
solution. This provides a means of differen-
tiating between the fluorescence from the
vitamin and that from possible interfering
substances. When applied to pharmaceutical pre-
parations, beverages and special dietary foods,
72 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

the method shows a high degree of specificity.

No interfering substances were found (32).

A rapid colorimetric determination of ascorbic

acid (10 to 200 mg per 100 g) with the sodium
salt of 2,6-dichlorophenol-indophenol was
studied. An amyl acetate extract of 2,6-
dichlorophenol-indophenol has max. absorption
at 525 nm the extinction of which remains
unchanged for 4 hrs. The sample is extracted
with 2 per cent metaphosphoric acid and
diluted to produce a 0 . 4 to 1.0 mg per cent
solution of ascorbic acid. 2,6-dichlorophenol-
indophenol solution (5 ml in water) is added
to the sample solution which is rapidly ex-
tracted with amyl acetate for the extinction
to be measured. Another 5-ml portion of the
2,6-dichlorophenol-indophenol solution is
added to metaphosphoric acid and similarly
treated. The amount of ascorbic acid is
determined from the difference in the extinc-
tion of the two extracts, which is propor-.
tional to the concentration of ascorbic acid
up to 4 mg per 100 ml ( 3 3 ) .
Other method depends on the reduction of
ferric chloride by ascorbic acid and the
colorimetric determination of the ferric
chloride by means of reaction with aa-dipy-
ridly; the reaction is carried out in the
presence of phosphoric acid to eliminate in-
terference by reduction. A solution (1 ml)
containing N 0.02 mg of ascorbic acid is tre-
ted with 0.3 ml of 85 per cent. Phosphoric
acid to give a pH of 1 to 2, 5 ml of 0.5 per
cent aqueous aa-dipyridly solution and 1 ml of
1 per cent ferric chloride. The method has
been applied to orange juice, honey and urine;
it is sensitive to 5 lJg of ascorbic acid per
ml ( 3 4 ) .
A direct method for the determination of vita-
min C without removal of the reagent, 2,4-
dinitrophenylhydrazine, is applied to blood
and urine. The ascorbic acid content i s calcu-
lated from a chart (35).
Ascorbic acid can be semi-quantitatively
estimated with the comparison method after
color reaction with molybdophosphoric acid.
ASCORBIC ACID 73

The red zone of dehydroascorbic acid-DNP is

scraped off, eluted with 85% sulphuric acid,
filtered or, better, centrifuged, and the
light absorption of the solution measured at
520 to 525 nm against water as blank. The
analysis result is worked out with the help of
a standard solution which is treated identi-
cally ( 9 ) .

9.3 Turbidimetric Method

This method (36) is used €or the determination o f
ascorbic acid in foods, by the reaction of selenious
acid with ascorbic acid and stannous ions at low pH
and room temperature.

9.4 Chromatographic Methods

9.4.1 Paper Chromatography

Methods used €or the separation of ascorbic

acid by paper chromatography are shown in
Table 5 ( p . 21).

9.4.2 Gas-Liquid Chromatography

Gerstl and Ranf€t (42) extracted food with

metaphosphoric acid, separted the ascorbic
acid on a column of cellulose and formed the
trimethysilyl ether derivative by reaction
with N.0-bis-(trimethylsily) acetamide before
chromatographing on a column of Gas Q contain-
ing 3% SE-30.
9.4.3 High Pressure Liquid Chromatography

Packla and Kissinger ( 4 3 ) , used a strong anion

resin and elution with pH 4.75 buffer solution,
has been applied to the determination of
ascorbic acid in milk products, baby foods,
fruit juice concentrates, whole fruits and
fortified cereals. The procedure was not
directly suitable for measuring dehydroascorbic
acid. However, sood et a1 (44) used HPLC in
 TABLE - 5 Methods used for separating ascorbic acid by paper chromatography
P
- -- -~ ~ ~~~ ~ ~

Chromatogram Solvent System Rf Value Reagent Application Reference

Paper Chro- 1) n-butanol saturated 2,6-dichlorophenol- Plant and 37

matography. with water + oxalic indophenol with sub- animal
sequent colorimetry. tissues.
2) Phenol saturated with
water + oxalic acid.
Paper (Sch- 1) 50% Methanol. 0.7-0.8 1) 1% silver-nitrate Lemon juice 38
leicher and solution in 10% white,red and
Schull No. Aqueous ammonia. black-currents.
602). 2) Wat er-n-butano1- 0.6-0.7 2 ) 0.005N iodine solu- tomatoes and
glacial acetic acid tion in 0,04% starch green pappers.
(50:40:14:5) so 1ut ion.
3) 0.04% aqueous solu-
tion of 2,6-dichloro-
phenol-indophenol.
4) U.V.
Paper Chro- butanol-acetic acid- 0.36 ammonium-molybdate solu- 39
matography. water (4:1 :5) tion.

Paper Chro- n-butanol-acetic acid- Alkaline tetrazolium Method f o r de- 40

matography. water (4:l:S) salts. tection of lfpg
quantity.
ASCORBIC ACID 75

the reverse phase ion-pairing mode to deter-

mine ascorbic acid in food with tridecyl ammo-
nium formate as counter-ion.

9.5 Enzymatic Method

A new enzymatic method based on the oxidation of

the ascorbic acid to dehydroascorbic acid with
ascorbic oxidase permit assaying for ascorbic acid
and dehydroascorbic acids in vegetable extracts (45).

9.6 Polarographic Method

Ascorbic acid can be analysed in a variety of mix-
tures and multivitamins preparations by cathode ray
reverse sweep polarography (46).

9.7 Chronometric Method

The reduction oxidation couple between ascorbic acid

oxidation and reduction of the semiquinoid form p-
phenylenediaimine is the basis of a new chronometric
assay of vitamin C.
Ascorbic acid interrupts formation of the colored
product by coupling its oxidation to reduction of
the semiquirioid proceeds to dye form (47).
76 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

10. References

1. G.C.G. Grasselli and N.M. Ritchey "Atlas of Spectral Data

and Physical Constants for Organic Compounds" 2nd Ed. ,
Vol. 2, CRC Press Inc., Cleveland, Ohio, 1975.
2. R.W. Herbert, E.L. Hirst, E.G.U. Percival, R.J.W. Reynolds
and Smith, J. Chem. SOC., 1270 (1933).
3. J.E. Hoover "Remington's Pharmaceutical Sciences", - 15th
Ed., Mack Publishing Company, U.S.A., (1975).
4. M. Windholz "The Merck Index" 9th Ed. Merck and Co., Inc.,
Rahaway, U.S.A. (1976).
5. "The British Pharmacopeia", Her Majesty's Stationary
Office, Cambridge, (1973).
6. "The British Pharmacopeia", Her Majesty's Stationary
Office, Cambridge, (1980).
7. "The United States Pharmacopeia", XX, Mack Publishing
Co., Easton, Pa (1980).
8. R.C.R. Barreto, Rev. Quim. Ind., Rio de Janeiro, - 24, 13,
(1955). Through, Anal. Abstract , 3, 567 (1956).
9. E. Stahl , "Thin Layer ChromatograpFy", 2nd Ed. , Springer-
Verlag., p. 304, (1966).
10. C.J. Pouchert and J.R. Campbell "The Aldrich Library of NMR
.
and Spectra" Vol XI (1974).
11. "High Resolution NMR Spectra Catalog", Varian Analytical
Instrument Division, Vol. 2, The National Press, U.S.A.
(1963) .
12. D.T. Sawyer and J.R. Brannan, Anal. Chem., 38 (2), 192,
(1966).
13. S . Berger, Tetrahedron, 33, 1587 (1977).
14. R.D. King "Developments in Food Analysis Technique-1"
Applied Science Publishers, 18 (1978).
15. C.B. Bourgeois, P.R. Mainuy, R. George and A.M. Czornomaz,
Analusis, 2, 556 (1973). Through Reference 14.
16. V.G. Randall, E.L., Pippen, A.L. Potter and R.M. McCready,
J. Fd. Sci., 40, 894. Through Reference 14.
17. G, Enachescu,%al. Inst. Cerc. Agron. Romn., 22, 463,
(1955) . Through Anal. Abstract , 3, 3474 (1956):
18. T.A. Geissman, "Principles of Organic Chemistry", 3rd. Ed.,
W.H. Freeman and Company, p. 497, (1968).
19. T. Reichstein and A. Grussner, tlelv. Chim. Acta., -17, 311,
(1934).
20. J. Bakke and 0. Theander Chemical Comm. , 175 (1971).
21. M. Luckner "Secondary Metabolism in Plants and Animals",
Science Paperbacks, p. 74, (1977).
22. E.G.C. Clarke "Isolation and Identification of Drugs",
The Pharmaceutical Press, London, (1971).
ASCORBIC ACID 77

23. T. Masaru, W. Sanae, M. Katsuko, T. Sachiko, F. Akji

(Biochem. Lab. Kagawa Nutr. Coll., Tokyo, Japan). Vila-
mins 45(3), 136, 1974, fJapan). Through
24. C.D. Wilson, 0. Gisvold and R . F . Doerge "Textbook o f Orga-
nic Medicinal and Pharmaceutical Chemistry", 7th Ed.,
J.B. Lippincott Co., Philadelphia, (1977).
25. E . Piyanowski, Prezem. Rony Spozywezy, 11,410 (1954).
26. G.A. Vaisman and S.G. Rozhntskaya Aptechnoc Delo. (3),
16. Through: Anal. Abstract, 2, 563 (1955).
27. S.R. Mohanty, K.R.K. Rao and L.V. Kannan, Anal. Chim.
Acta, 14 (6) 587 (1956). Through: Anal. Abstract, 2, 347,
(1956);
28. G.S. Deshmukh and M.G. Bapat, Z. Anal. Chem. 145(4), 256
(1955).
29. (a) M. Schmall, C.W. Pifer and E.G. Wollish, Anal. Chem.
25(10), 1486 (1953). Through Anal. Abstract, 1,370,
-
(1954).
(b) M.H. Hashmi, M.A. Shahid, M.A. Akhtar and N.A. Chugtani,
Mikrochim Acta, 5, 901 (1973).
30. H.. Freytag, Z. anal. Chem., 139(4), 263, 1953. Through.
Anal. Abstract, 1, 371, (1954).
31. J.A. Owen and B.Iggo, Biochem. J., 62(4), 675 (1956).
32. C. Mike, J. Deutch and C.E. Weeks, J. Assoc. Office Agr.
Chemists, 48(6), 1248 (1965). Through Chem. Abstract, 64,
72840 (1966).
33* H. Imai and T. Fugitani J. Chem. SOC., Japan, Pure Chem.
Sect., s(11), 1212 (1955). Through: Anal. Abstract 2,
2567 (1956).
34. M.X. Sullivan and H.G.N. Clarke, J. Ass. Off. Agric. Chem.,
38(2), 514 (1955). Through: Anal. Abstract, 2, 258 (1956).
-
35. J.H. Roc and C.A. Kuether, J. Biol. Chem. 147, 399 (1943).
Through: Chem. Abstract, 37, 31185 (1943).
36. W.J. Ralls, J. Agric. Food Chem. 23(3), 609, (1975).
Through: Chem. Abstract, 83, 41610 F (1975).
37. Yu-Tuan Cheng., F.A. Isherwood and L.W. Mapson, Biochem.
J., 55(5), 821 (1953). Through: Anal. Abstract, I, 372,
(1954).
38. V. Sanda Ceskosl. Farmac., 2(3), 79 (1954). Through: Anal.
Abstract, 2, 2563, (1955).
39. W. Hermann, R. Strohecker and F. Matt., Z. Lebensmitt. Un-
tersuch. U. Forsch., 97, 263, (1953). Through: Anal.
Abstract, 1, 369, 1954.
40. 2. Padre, E. Smid and V. Sicho, Naturwissenschaften, 42(8) ,
210, (1955). Through: Anal. Abstract, 2, 853 (1956).
41. J.E. Schlack, J. Ass. Office, Analyt. Chemist., 57, 1346,
(1974). Through: Reference 14.
42. R. Gerstl and K. Ranfft, Z. Lebensmitt-elunters. U. Forsch.,
-
154, 12, (1974). Through Reference 14.
78 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

43. L . A . Packla and P.T. Kissinger, Anal. Chem., 48, 3 6 4 ,

( 1 9 7 6 ) . Through Reference 1 4 .
4 4 . S.P. Sood, L.E. Sartori, D.P. Wittmer and W.G. Haney,
Anal., Chin., 4 8 , 796, ( 1 9 7 6 ) . Through Reference 1 4 .
4 5 . A. Marchesini, F . , Montauori, D. Muffats, D. Maestri, J.
Food Sci., 3 9 ( 3 ) , 5 6 8 , ( 1 9 7 4 ) . Through Chem. Abstract,
8 2 , 2717 F ( 1 9 7 5 ) .
4 6 . K S . Owen, F.W. Franklin, J. Food Technol., E ( 3 ) , 2 6 3 ,
( 1 9 7 5 ) . Through Chem. Abstract, 83, 1 3 0 1 0 2 J ( 1 9 7 5 ) .
4 7 . B. Roe, J . H . Bruemmer, Proc. Fla. State Hortic. SOC.,
87, 210, ( 1 9 7 5 ) . Through Chem. Abstract, 83, 130100 G
-
(1975).