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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
""-H-CJ7 I
+ H,C.CI'


- O=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
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

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
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
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.
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 volume of orange juice used should be increased to 10 ml, or further
one of the ahove analytical reagents. A roster system was if necessary. The titration should he repeated until consistent results
provided to ensure tbat students attempted both of the ti- are ohtained.
trimetric ~rocedures. Statistical Analysis of Results
The an&& may be conveniently divided into three stages, Statistical analysis occupied the fourth laboratory session
these being preparation of sample, standardization of oxi-
of the project. For convenience, the dara ohtained on a single
dizing age&;and titration procedure. day for a panirular brand of juice was classified as a "set" and
While it is possible to perform the titration directly on or-
this set was divided into twosuhsets, each containing thr re-
ange juice, the color of the juice can sometimes obscure the
sults ohtained by oneof the twoanalytical proredures. Groups
endpoint. For this reason, it is advisable to either filter off the
of three students were allocated a subset and asked to perform
pulpy components of the juice or to extract the orange coloring
a divergent data Q test and to calculatr mean and standard
matter using ether. Orange juice is very difficult to filter be-
deviation of the results. Next, the studentsselected a pair of
cause the suspended matter quickly clogs the pores of the filter suhscts and performed an F test and Student's I test' to as-
paper; use of a filter aid such as celite or supercel is therefore
certain whether any significant difference existed between the
necessary. chosen pair of subsets. An optional section requiring perfor-
The stock solutions of NBS ( 4 . 0 0 1 M ) and 2,6 DPIP
mance of a Student's t test on the results obtained by filtration
(-0.001 M ) are unstable and it is therefore necessary to and those obtained by ether extraction was also included. The
standardize them before use. This standardization may he students were asked to present the results graphically and to
accomplished using many common primary standards; how-
discuss in detail all possible sources of error.
ever it is more convenient to use pure ascorhic acid as stan-
dard. Results
Directions The results of the project. indicated that under the condi-
tions of storace and aeratim used, tinned orange juice (which
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 is pasteurized and contains no preservative) lost 36% of its
the oxalic acid has dissolved. The purpose of the oxalic acid is to ascorbic acid content over a period of 14 days, while a second
stabilize the ascorbic acid in the juice, which could otherwise he oxi- variety of juice which used sorbic acid as preservative lost 21%
dized during the filtration process, and to acidify the titration solu- of its ascorbic acid content over a similar period. The other
tion. brands of juice examined, all of which used henzoic acid or
Add sufficientfilter aid to the orange juice to form a thin paste and sulfur dioxide (or a combination of the two) as preservative,
filter this paste through a Buchner funnel into a clean, dry flask. The showed no statisticallv sienificant change in ascorhic acid
filtration should proceed rapidly, but if this is not the case, remove content during 14 daysof storage. These &wlts indicate that
the juice and add a little more filter aid. The resulting filteredsolution for a marked decrease in ascorhic acid content to he observed,
should be only pale orange in color and quite transparent. This so- tinned juices or those using sorbic acid as preservative should
lution is now ready for analysis.
An alternativemethod of sample preparation is to extract the or- be selected as samples.
anee enlor into ether. Take ahaut 30-40 ml of oranee .. .iuice. add 0.2 e The results of the NBS and 2,6 DPIP methods were com-
rold <mal~racid and swirl u n t i l thr oxsl~cacidhasdissolvrd. Trnnder parable and showed no significant difference a t the 99% con-
this sulution to a separating iunnrl nnd add n l s u t 20 ml rrhrr fr8.m fidence level. However, the precision of the 2,6 DPIP method
a measuring cylinder (CARE! ether is highly inflammable-ensure was generally worse than tbat for the NBS method, as shown
that there are no naked flames in the laboratory).Gently shake the by the average relative standard deviations (6.5% for NBS,
funnel-too much shaking will result in the farmationofan emulsion 7.7% for 2,6 DPIP). This difference in precision can be at-
which will take a long time to separate. The orange color will be tributed to the difficulty in detecting the endpoint of the 2,6
transferred to the ether (uooer) laver and most of the o . u.l will
~ tend
DPIP titration.
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 The only problem encountered by the students was failure
pulp in the separating funnrl 'The nquwut phaw I. no\v rend) for to perform the titrations rapidly-slow titration permits slow
analysis. acting reducing agents present in the juice to interfere a t the
Stondardimtion of Oxidizing Agent. Weigh accurately about 50 endpoint, causing gradual reversal of the color change. The
mg of ascorhic acid and quantitatively transfer it to a 250-ml volu- values obtained for the ascorbic acid content of those iuices
metric flask containing about 2gsolid oxalicacid. Fill the flask to the containing sulfur dioxide required correction for interference
mark and shakegently until all solid has dissolved. This solution may bv this nreservative. Correction was performed mathemati-
he used to standardize the N-bromosuccinimideand 2.6 DPIP solu- callv inbur project sinrt. the manufaFturers ohliged by pro-
tions provided using the procedures described below.
NBS; Add a 25-ml aliquot of the standard ascorbic acid solution vidine the rlass w t h the concentration of SO! present in the
prepared above to a conical flask containing 5 ml of 4% potassium juices; if this information is not available, t h i n SO? can he
iodide solution, 2 ml of 10%acetic acid solution, and 3 drops of starch masked in the titration procedure by addition of 1 ml 2 M
indicator. Dilute with about 30 ml of distilled water and titrate with HzS04 and 1 ml of 40% formaldehyde solution to each 5-ml
the NBS solution ~rovided.The endpoint is marked by the appear- ~ ~
aliquot of juice prior to titration. Excess formaldehyde does
ance of a permanent blue color. not react with 2,6 DPIP or NBS.
2,6DPIP: Add a 25-ml aliquot of the standard ascorhic acid solution This project proved to he the most popular experiment of
prepared above to a conical flask,dilute with about 30 ml of distilled the c o m e and the degree of student interest and involvement
water, and titrate with the 2.6 DPIP solution provided. The endpoint was very rewarding.
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 Youmans, H. L., "Statistics for Chemistry," Charles E. Merrill
ascorbic acid solution. If a titre of less than 10 rnl is obtained, the Publishing Co., Columbus, Ohio, 1973, pp. 100-107.

Volume 54, Number 4 March 1977 1 193