Paul Haddad

I Vitamin C Content of Commercial Orange

Australian National University
P.O. Box 4 Juices
Canberra, A.C.T.. 2600, Australia

An analytical project

It has become customary at this University for the first year, (Oxidation)
nonchemistry major students, to conduct a survey involving
the analysis of a consumer item and to perform a detailed
statistical evaluation of the results.
The project for 1975 occupied four 3%-hr laboratory ses-
sions and involved a studs of the magnitude and stability of
the ascorbic acid conten<of commercial orange juices. This
studs had tu.0 aims: firstly t o confirm that newly purchased
juicecontained sufficient ascorbic acid to meet government
standards (at present 40 mg/100 ml of juice), and secondly to
establish the rate of aerial oxidation of this ascorbic acid when
HO-c
11
""-H-CJ7 I
0
H,C-~\
+ H,C.CI'
/O

N-Br

N"
n
- O=C

H-C
the juice was stored in a refrigerator. Students were interested
in ascertaining if the juice represented a suitable source of I
Vitamin C after one or two weeks of storage.
The Proled
HO-C-H
I
I HO-C-H
I
In view of the background of the students, it was considered
appropriate to select a redox titrimetric procedure, and the
~~~~~~~ ~~~~
.
most satisfactorv methods for determination of ascorbic acid
~~~

in orange juice were round to be those using the reagents 2,fi

dichloroohenolindoohenol 12.6 DPIP)l and N-hromosucci-
nimide (NBS).2 In order to provide comparison data for use
in the statistical analysis section of the project, both of these
reagents were used concurrently.
The reaction of 2,6 DPIP with ascorbic acid is shown L-ascorbic N-bromosuceinimide L-dehydro
below. acid ascorbic acid
(Reduction)

Use of a starch iodide indicator provides a color change of

0 0 colorless to blue a t the endpoint.
I1 II Experlmenlal
Samples and Reagents
HO-C
Five brands of juice were used for the project and 6 1of each
were ourchased (this was sufficient for 150 students). In se-
lection of the hrands, n)nsiderari~mwas given to tnedifferent
oresewatives allowed sulfur dioxide, sorbir acid. and benzoic
Hcid) and also to the different formsof pa~kage~availahle, so
HO-C-H HO-C-H that the samoles chosen were reoresentative of the ranae - sold
I I on the market. In addition, to minimize sample variation, the
juice was obtained in 1-1 containers and then combined to
produce the volume required for the project.
Each brand of juice was stored in a separate sealed plastic
container at 0C and was lightly aerated daily by shaking and
(Oxidation)
pouring the contents into another container. This process was
Gascorbic 2,6-dichlorophenol L-dehydro intended to simulate normal household treatment of orange
acid indophenol ascorbic acid juice. Two liters of this juice were supplied on each practical
2,6 DPIP is blue in neutral or alkaline solution and red in acid day attended by the group which had been allotted that par-
solution while its reduced form is colorless. Thus when ti- ticular brand for analysis.
trating an acidic solution of ascorbic acid against blue 2,6 2,6 DPIP was obtained from BDH Chemicals Limited and
DPIP, the blue reagent will turn colorless while ascorbic acid a stock solution was prepared daily by dissolving 1.25 g of 2,6
is present, but when all the ascorbic acid has been consumed, DPIP and 1.05 g NaHC03 in 5 l distilled water. NBS was ob-
any excess 2,6 DPIP added will turn the solution pink, thereby tained from BDH Chemicals Limited and a solution con-
signifying the endpoint. taining 1.0 g NBS in 5 1 distilled water was prepared fresh
NBS reacts with ascorbic acid as follows daily. These quantities were sufficient for 30 students.
General
Gyorgy, R., and Pearson, W. N. (Editors),"The Vitamins", Vol.
VII, 2nd ed., Academic Press, New York, 1967, pp 27-51. Practical sessions were conducted every weekday and those
2 Barakat, M. Z., El-Waheb, M. F. A,, and El-Sadr, M. M., Anal. students attending on a particular day were allocated a brand
Chem., 27,536, (1955). of juice to analyze. Each group was further divided into two
192 1 Journal of ChemicalEducation
subgroups (of about 15 students) with each subgroup using
one of the ahove analytical reagents. A roster system was
provided to ensure tbat students attempted both of the ti-
trimetric ~rocedures.
The an&& may be conveniently divided into three stages,
these being preparation of sample, standardization of oxi-
dizing age&;and titration procedure.
While it is possible to perform the titration directly on or-
ange juice, the color of the juice can sometimes obscure the
endpoint. For this reason, it is advisable to either filter off the
pulpy components of the juice or to extract the orange coloring
matter using ether. Orange juice is very difficult to filter be-
cause the suspended matter quickly clogs the pores of the filter
paper; use of a filter aid such as celite or supercel is therefore
necessary.
The stock solutions of NBS ( 4 . 0 0 1 M ) and 2,6 DPIP
(-0.001 M ) are unstable and it is therefore necessary to
standardize them before use. This standardization may he
accomplished using many common primary standards; how-
ever it is more convenient to use pure ascorhic acid as stan-
dard.
Directions
Preparation of Samples. Take about 3040ml of orange juice, add
0.2 g solid o d i c acid (CARE! oxalie acid is poisonous),and swirl until
the oxalic acid has dissolved. The purpose of the oxalic acid is to
stabilize the ascorbic acid in the juice, which could otherwise he oxi-
dized during the filtration process, and to acidify the titration solu-
tion.
Add sufficientfilter aid to the orange juice to form a thin paste and
filter this paste through a Buchner funnel into a clean, dry flask. The
filtration should proceed rapidly, but if this is not the case, remove
the juice and add a little more filter aid. The resulting filteredsolution
should be only pale orange in color and quite transparent. This so-
lution is now ready for analysis.
An alternativemethod of sample preparation is to extract the or-
anee enlor into ether. Take ahaut 30-40 ml of oranee .. .iuice. add 0.2 e
rold <mal~racid and swirl u n t i l thr oxsl~cacidhasdissolvrd. Trnnder
this sulution to a separating iunnrl nnd add n l s u t 20 ml rrhrr fr8.m
a measuring cylinder (CARE! ether is highly inflammable-ensure
that there are no naked flames in the laboratory).Gently shake the
funnel-too much shaking will result in the farmationofan emulsion
which will take a long time to separate. The orange color will be
..
transferred to the ether (uooer) laver and most of the o . u.l will
~ tend
1 8 0 ndlect n t the ph:iie Irundar) Ibrrurw the a q u c w i and orgsnlc
layers. IJrein off the a q ~ e m 1 plmr
4 _wving8s mu( h a j pmihlr of the
pulp in the separating funnrl 'The nquwut phaw I. no\v rend) for
analysis.
Stondardimtion of Oxidizing Agent. Weigh accurately about 50
mg of ascorhic acid and quantitatively transfer it to a 250-ml volu-
metric flask containing about 2gsolid oxalicacid. Fill the flask to the
mark and shakegently until all solid has dissolved. This solution may
he used to standardize the N-bromosuccinimideand 2.6 DPIP solu-
tions provided using the procedures described below.
NBS; Add a 25-ml aliquot of the standard ascorbic acid solution
prepared above to a conical flask containing 5 ml of 4% potassium
iodide solution, 2 ml of 10%acetic acid solution, and 3 drops of starch
indicator. Dilute with about 30 ml of distilled water and titrate with
the NBS solution ~rovided.The endpoint is marked by the appear- ~ ~
ance of a permanent blue color.
2,6DPIP: Add a 25-ml aliquot of the standard ascorhic acid solution
prepared above to a conical flask,dilute with about 30 ml of distilled
water, and titrate with the 2.6 DPIP solution provided. The endpoint
is marked by the appearance of the first permanent pink color.
Titration Procedure; Using a 5-ml aliquot of prepared orange juice,
proceed as outlined above in "standardization of midizing agent" with
the orange juice aliquot replacing the 25-ml aliquot of standard
ascorbic acid solution. If a titre of less than 10 rnl is obtained, the
volume of orange juice used should be increased to 10 ml, or further
if necessary. The titration should he repeated until consistent results
are ohtained.
Statistical Analysis of Results
Statistical analysis occupied the fourth laboratory session
of the project. For convenience, the dara ohtained on a single
day for a panirular brand of juice was classified as a "set" and
this set was divided into twosuhsets, each containing thr re-
sults ohtained by oneof the twoanalytical proredures. Groups
of three students were allocated a subset and asked to perform
a divergent data Q test and to calculatr mean and standard
deviation of the results. Next, the studentsselected a pair of
suhscts and performed an F test and Student's I test' to as-
certain whether any significant difference existed between the
chosen pair of subsets. An optional section requiring perfor-
mance of a Student's t test on the results obtained by filtration
and those obtained by ether extraction was also included. The
students were asked to present the results graphically and to
discuss in detail all possible sources of error.
Results
The results of the project. indicated that under the condi-
tions of storace and aeratim used, tinned orange juice (which
is pasteurized and contains no preservative) lost 36% of its
ascorbic acid content over a period of 14 days, while a second
variety of juice which used sorbic acid as preservative lost 21%
of its ascorbic acid content over a similar period. The other
brands of juice examined, all of which used henzoic acid or
sulfur dioxide (or a combination of the two) as preservative,
showed no statisticallv sienificant change in ascorhic acid
content during 14 daysof storage. These &wlts indicate that
for a marked decrease in ascorhic acid content to he observed,
tinned juices or those using sorbic acid as preservative should
be selected as samples.
The results of the NBS and 2,6 DPIP methods were com-
parable and showed no significant difference a t the 99% con-
fidence level. However, the precision of the 2,6 DPIP method
was generally worse than tbat for the NBS method, as shown
by the average relative standard deviations (6.5% for NBS,
7.7% for 2,6 DPIP). This difference in precision can be at-
tributed to the difficulty in detecting the endpoint of the 2,6
DPIP titration.
The only problem encountered by the students was failure
to perform the titrations rapidly-slow titration permits slow
acting reducing agents present in the juice to interfere a t the
endpoint, causing gradual reversal of the color change. The
values obtained for the ascorbic acid content of those iuices
containing sulfur dioxide required correction for interference
bv this nreservative. Correction was performed mathemati-
callv inbur project sinrt. the manufaFturers ohliged by pro-
vidine the rlass w t h the concentration of SO! present in the
juices; if this information is not available, t h i n SO? can he
masked in the titration procedure by addition of 1 ml 2 M
HzS04 and 1 ml of 40% formaldehyde solution to each 5-ml
aliquot of juice prior to titration. Excess formaldehyde does
not react with 2,6 DPIP or NBS.
This project proved to he the most popular experiment of
the c o m e and the degree of student interest and involvement
was very rewarding.
Youmans, H. L., "Statistics for Chemistry," Charles E. Merrill
Publishing Co., Columbus, Ohio, 1973, pp. 100-107.
Volume 54, Number 4 March 1977 1 193