127, 178- 182 (1982)
A New Calorimetric
of Vitamin C Using Folin Phenol Reagent
Technique for the Estimation
S. K. JAGOTA AND H. M. DANI
Department of Biochemistry, Panjab University, Chandigarh-160 014, India
Received June 14, 1982
A new calorimetric technique for the estimation of ascorbic acid by using Folin phenol
reagent has been developed. The absorption maximum of the color developed by the interaction
of ascorbic acid with Folin reagent is 760 nm. The technique obeys the Beer-Lambert
to a concentration of 45 pg ascorbic acid as shown by the standard curve. The color developed
has been found to be stable up to 18 h. Recovery experiments showed that the technique is
almost 100% efficient. The development of the color is not obstructed by glucose, ghttathione,
bovine serum albumin, urea, cysteine, adenine, guanine, cytosine, uracil, sulfosalicylic acid,
thymol, or oxyhemoglobin, which are compounds suspected of interfering in routine analysis.
The technique is simple, quick, and efficient and can be employed for the estimation of ascorbic
acid in a wide variety of biological materials.
number of biological
used for its estimation
ylene blue, iodine,
During its titration
number of other reducing agents (stannous
sulfate, sulfydryl compounds,
sulfates, and reducing materials found in car-
amelized and fermented foods) interfere, re-
sulting in nonspecificity
Apart from this, the titrimetric
not be used for estimating
whole blood unless oxyhemoglobin
of ascorbic acid) is rendered inert by satu-
ration with carbon monoxide
method of Butler and Cushman
reduction as in the procedure developed by
Kuether and Roe (3). The titrimetric
ods are therefore not applicable
work. The calorimetric
ent involves reaction of dehydroascorbic
C is routinely estimated in a
materials and is of tre-
importance. The methods
are based on its oxi-
properties. The procedures
of an acid extract with meth-
or other oxidizing
of the reaction.
ascorbic acid in
as in the
(2) or by
method used at pres-
hydrazine to form an
osazone which reacts with strong sulfuric
acid to give a red color (4). The method
quite laborious and time
needs a minimum of about 4 h for estima-
Folin reagent, an oxidizing
blue color on reduction
estimation of proteins at pH 10 (5). During
our studies on the interaction
with microsomes (6), we observed that ascor-
bic acid interferes with protein estimation,
and on the basis of this we developed a new
technique for the estimation
by the use of Folin-Ciocalteu
acidic range of pH. The technique is simple,
quick, and efficient and can be used for the
estimation of ascorbic acid in blood without
prior conversion to its dehydro form.
agent, gives a
and is used for the
of ascorbic acid
of ascorbic acid
reagent in an
MATERIALS AND METHODS
purchased from CSIR Centre for Biochemi-
cals, New Delhi, India. Cytosine and guanine
were purchased from SRL, Bombay. Glucose
(A.R.), urea (A.R.), adenine, and uracil were
All rights of reproduction
0 1982 by Academic
in any form reserved.
VITAMIN C ESTIMATION BY FOLIN REAGENT
0.0 a . . I I I I I . t
400 LB0 560 640 720
900 860 960
FIG. 1. Absorption spectra of the color developed by
the interaction of ascorbic acid with Folin reagent.
the products of B.D.H.
min (A.R.) and sulfosalicylic acid (A.R.) were
obtained from Sigma Chemical
Thymol (A.R.) and ascorbic acid (G.R.) were
bought from S. Merck. Glutathione
chased from Loba-Chemie.
icals (except of A.R. and G.R. grades) were
of reagent grade.
Bovine serum albu-
All other chem-
1. 10% Trichloroacetic
reagent of 2.0 M concentration
IO-fold with double-distilled
3. Ascorbic acid standard solution
100 pg ascorbic acid/ml.
Procedure. To 0.2 ml of tissue homoge-
nate, lemon juice, blood
blood 0.8 ml of 10% trichioroacetic
added. After vigorous shaking the tubes were
kept in an ice bath for 5 min and centrifuged
at 3000 rpm for another 5 min. For the es-
timation of ascorbic acid, 0.2-0.5 ml of the
above extract was used. The extract was di-
luted to 2.0 ml using double-distilled
and after 0.2 ml of diluted Folin reagent was
added to the extract, the tubes were vigor-
ously shaken. After 10 min the absorbance
of the blue color developed was measured in
a Bausch & Lomb Spectronic-70
serum, or whole
by taking 0.05-0.7
of ascorbic acid in water (5-70 pg) and es-
timating as above. For checking the possible
interference of various compounds,
each compound was added to the assay mix-
ture before estimation.
A standard curve was prepared
ml of standard solutions
50 pg of
RESULTS AND DISCUSSION
between 380 and 950 nm is depicted in Fig.
1. Absorption maximum
at 760 nm.
The standard curve of ascorbic acid is
shown in Fig. 2. The curve is linear up to an
ascorbic acid concentration
beyond this concentration,
not seem to follow the Beer-Lambert
Data on stability of the color developed
are presented in Table 1. Maximum
developed after 10 min and then remains
stable up to 18 h.
Effect of pH on the intensity of the color
developed was studied in the acidic range
(pH 1 .O-7.0) and it was concluded that vari-
ations of pH in this range have no effect on
the color complex (Table 2). As Folin reagent
reacts specifically with ascorbic acid in a
broad pH range, there does not seem to be
any necessity for developing
for maintaining any specific pH. In the acidic
pH range Folin reagent is known to have
of the color developed
of the color occurs
of 45 pg, but
the method does
a buffer system
I I I 1 I 8
0 50 100 15-O 20.0 25.0 300 35.0 40.0 L5.0
ASCORBIC ACID CONCENlRATION(,q)
FIG. 2. Standard curve for ascorbic acid estimation.
JAGOTA AND DAN1
AT ROOM TEMPERATURE
Optical density at 760 nm at
different time intervals’ Ascorbic acid
(a) 10 min 35 min 70 min 18 h
0.2 M Buffer system
n Tris-HCI buffer of different molarities (0.1 to 2.0 M)
at pH 1.0 to 7.0 was also tested, and no significant dif-
ferences in absorbance at 760 nm were recorded.
tightly with aluminum foil and left at room temperature
for the time intervals indicated.
color development the tubes were covered
reducing agents like ascorbic acid can reduce
this reagent, the chances of interference by
other mild reducing agents are highly de-
The efficiency of this method was checked
by performing recovery experiments in three
reactivity, and because only strong
different sources (lemon juice, liver homog-
enate, and blood serum). These experiments
were done to reveal the chances of interfer-
ence in the interaction
and Folin reagent by any other reducing
agents possibly present in the biological
terials. The data presented in Table 3 indicate
that ascorbic acid is almost completely
covered even when a fixed quantity
vitamin is added to different quantities of the
between ascorbic acid
at 760 nm
Concentrations of ascorbic acid (pg/ml)
Supplemented with 200 pg
ascorbic acid/ml sample
a Samples of different concentrations in each case were divided into two equal parts, and ascorbic acid (200
&ml) was added to one part and the second part served as control. Extraction and the estimation of ascorbic acid
were performed as described under Materials and Methods.
VITAMIN C ESTIMATION BY FOLIN REAGENT
ABSORBANCE OF COLORS DEVELOPED ON INCUBATION
OF FOLIN REAGENT WITH COMPOUNDS LIKELY TO
INTERFERE WITH ASCORBIC ACID ESTIMATION
COMPARISON OF ASCORBIC ACID CONTENT IN VAR-
IOUS BIOLOGICAL MATERIALS
LIN PHENOL REAGENT AND
Absorbance (760 nm)
after I5-min incubation Compounds”
Bovine serum albumin
a Fifty micrograms of each compound was added to
an assay mixture in place of ascorbic acid.
Values in literature
25.2 + 1.11”
8.56 + 0.20”
1.44 k 0.16b
1.65 kO.8’ (II)
I.16 + 0.23b
0.75 to 1.56 (12)
100 g tissue.
100 ml of serum or blood.
to be quite efficient.
Folin reagent can react with a number of
biological materials such as guanine,
thine (7,8), and most of the phenols (9) in
alkaline media (pH 10.0). Thymol
fosalicylic acid also interfere with the reac-
tion of this reagent with proteins. Similarly,
a number of substances interfere with ascor-
bic acid estimation by the existing
niques. Therefore, possible interference of a
number of compounds in this technique were
screened up to 50 pg of concentration
assay mixtures (Table 4). Glutathione
duced), cysteine, glucose, bovine serum al-
bumin, urea, adenine, uracil, thymine,
nine, cytosine, oxyhemoglobin,
fosalicylic acid, and trichloroacetic
not develop color complex with absorption
maximum of 760 nm and therefore could
not interfere in the ascorbic acid estimation.
Complete absence of the interference
even those substances which interfere in pro-
tein estimation by Folin reagent may be due
to either dilution of the Folin reagent or to
acidic pH of the media. It is well known that
in acidic pH the dissociation
Therefore, the method seems
of yellow color of phosphomolybdate
decreases the reactivity
Only strong reductants like ascorbic acid can
react with Folin reagent under these condi-
tions, and any interference by other possible
substances is eliminated.
To compare the accuracy of our technique
with already available methods, ascorbic acid
estimations were made in rat liver, kidney,
testis, pancreas, spleen, and serum and hu-
man whole blood. The comparative
obtained with our method and those avail-
able in literature employing
mentioned materials are presented in Table
5. Our method provides values of ascorbic
acid in all these materials that are very close
to those reported in literature.
The technique presented in this paper for
vitamin C estimation
quick, efficient, and simple for a wide variety
of biological materials, particularly
ical laboratories which can use whole blood
in place of plasma as the starting material.
leads to the disappearance
reagent. of Folin
in case of above
seems to be quite
The authors are grateful to UGC, New Delhi, India,
for awarding a research fellowship to S. K. Jagota.
JAGOTA AND DANI
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