PROCEDURES

Procedures for the determination of vitamin C in foods have continued to

draw the attention of analysts. Recent reviews in this subject include those of

Christie and Wiggins (1978), Cooke and Moxon (1981), Sauberlich et al. (1982)

and Pachla et al.(1985). The complex biological relationship between the

compound(s) possessing vitamin C activity, as well as the chemical similarity of

these compounds to others that are inactive, has made the existence of a single,

simple, and specific method close-to impossible (Sauberlich et al., 1982). Thus,

there has been a proliferation of methods developed, resulting in hundreds and

possibly thousands of papers reported. However, they mostly describe minor

variations on a fairly limited range of possible procedures, based essentially on

the chemical reactions of ascorbic acid and DHAA(Cooke and Moxon,1981).

The Association of Official Analytical Chemists (AOAC) has designated

two official methods for the determination of vitamin C: the dye-titration

method and the microfluorometric method (AOAC, 1984). The former method

makes use of the reducing power of the vitamin, and employs 2, 6-

dichlorophenolindophenol (DCIP) as the redox indicator for the determination

of ascorbic acid. In the micro-fluorometric method, ascorbic acid is oxidized

with active carbon to dehydroascorbic acid to form a fluorescent quinoxaline

compound which is measured in a fluorometer.

DETERMINATION OF VITAMIN C IN FRESH FRUITS AND

VEGETABLES USING THE DYE-TITRATION AND

MICROFLUOROMETRIC METHOD

Determination of Vitamin C in Fresh Fruits and Vegetables Using the

Dye-titration and Microfluorometric Method has been Studied 2. The vitamin C

content of 19 types of fresh fruits and 24 vegetables was determined by the

official AOAC methods of dye-titration and microfluorometry. As expected,

values obtained by the latter method, which estimated ascorbic acid plus

dehydroasorbic acid (DHAA), were clearly higher than those given by the

titration method, which determined only ascorbic acid. There were considerable

differences in the values obtained by the two methods, depending on the

concentration of DHAA in the foods. Larger differences were obtained for the

vegetables. The mean recovery value obtained by the dye-titration method was

significantly higher than that given by the fluorometric method (p<0.01).

Reproducibility studies showed that the two methods did not give significantly

different variances (p<0.05). If only ascorbic acid values were required, the

titration procedure would give good results, and it may be carried out rapidly

using simple laboratory equipment. If a fluorometer was available, total vitamin

C values, which would be more useful from the nutritional point of view, could

be determined.
DETERMINATION OF ASCORBIC ACID (VITAMIN C) IN

COMMERCIAL TABLET BY IODOMETRIC TRTRATION

Determination of Ascorbic acid (Vitamin C) in Commercial Tablet by

Iodometric Trtration has been Studied3. A 0.010 M solution of KIO3 was

prepared as a primary standard and titrated by an iodometric procedure against a

solution of sodium thiosulfate. In this way, the concentration of the thiouslfate

solution was determined to be 0.075 M. Using 50.00 mL of this standard iodate

solution, a corresponding quantity of I3- was allowed to react with ascorbic acid

present in a known quantity of a crushed vitamin C tablet. By determining the

left over amount of I3-, the amount of ascorbic acid present in vitamin C tables

was determined to be 59.30% w/w.

EFFECT OF PRESSURE COOKING ON VITAMIN C CONTENT OF

VEGETABLES

Effect of pressure cooking on vitamin c content of vegetables has been

studied4. A comparison has been made of the effect upon the retention of

vitamin C in 10 different vegetables when cooked in a modern pressure

saucepan and in an ordinary saucepan according to the recommended methods.

On the average the retention was higher in vegetables cooked in the pressure

cooker. The method of estimation used was that of Harris and Olliver (1942).
AN ENZYMIC METHOD FOR THE ESTIMATION OF TRUE

VITAMIN C*

An enzymic method for the estimation of true vitamin c * has been

studied5. The quantitative determination of vitamin C by chemical methods has

been the subject of a number of recent investigations. Biologic assay is usually

more difficult, more costly, and necessarily much slower. It has been more

accurate, however, up to the present time. The chief inaccuracy of the

volumetric method of Tillmans and associates (1) is that certain other

constituents of plant and animal tissues reduce the dye, since their reduction

potential is lower that that of the indicator. The recent isolation (2, 3) of a

powerful and specific ascorbic acid oxidase afforded us an opportunity of

devising a method for the determination of true vitamin C content. In brief, this

consists of determining by a suitable procedure the reducing power of tissue

extract before and after the action of the enzyme. The difference, of course, is a

measure of the amount of vitamin C present.

All tissues, juices, and body fluids cannot be treated alike. If it is

suspected that some of the vitamin is present in the oxidized (non-reducing)

form, it may be reduced. This is accomplished in the course of the preliminary

mercuric acetate purification procedure (4), which is often essential for accurate

results. We are therefore reporting the technique as we have applied it to several

typical foodstuffs. The reasons for the variations are usually self evident and

their applications to other materials will be easily appreciated.

 TABLE I

Analysis for Vitamin C per Gm. Of Substance

Total Reduction True

reduction not due to ascorbic

ascorbic acid

acid
mg. mg. mg.

Orange juice ………………………… 0.52 0.05 0.47

Lemon juice …………………………. 0.61 0.00 0.61

Tangerine juice ……………………… 0.50 Trace 0.50

Grapefruit juice ……………………... 0.62 0.00 0.62

Hubbard squash* …………………… 0.00 0.015

Tea* …………………………………. 0.48 (?) 0.26 0.22

No reduction after
Beer* …………………………………
mercuric acetate

treatment
Mick (grade B, pasteurized) per 10 0.00 0.07

cc …………………………………...... 0.07

* Prolonged bleaching occurred until tannins, pigments, etc., were

removed by treatment with mercuric acetate.

SPECTROPHOTOMETRIC DETERMINATION OF VITAMIN C IN

CITRUS FRUITS USING PERI-NAPHTHINDAN-2, 3, 4-TRIONE

 spectrophotometric determination of vitamin c in citrus fruits using peri-

naphthindan-2, 3, 4-trione has been studied6. A spectrophotometric method for

the determination of vitamin c in lemon, orange, and grapefruit juices is

described. It is based on the reaction with peri-naphthindan-2, 3, 4-trione

hydrate at pH 3 to give dihydroxy-peri-naphthindenone which is dissolved in

methanol and measured at 460 nm. The method shows good precision and its

accuracy compares favourably with the standard titrimetric procedure of the

U.S.P. No interferences are caused by dehydroascorbic acid as well as many

other juice ingredients.

APPLICATION OF DIFFERENTIAL PULSE POLAROGRAPHY TO

THE ASSAY OF ASCORBIC ACID

Application of differential pulse polarography to the assay of ascorbic

acid has been studied7. A polarographic method for the determination of

ascorbic acid in pharmaceutical preparations and fruit juices is described. The

method is based on the oxidation of ascorbic acid by iodine, and the resulting

iodide, after removal of excess iodine, is oxidized with bromine to iodate which

is measured polarographically. The method enables analysis of solutions as

dilute as 1.7 X 10-6 M ascorbic acid.

THALLIC PERCHLORATE IN ACID MEDIUM AS A REAGENT FOR

OXIDIMETRIC DETERMINATION OF ASCORBIC ACID IN DRUGS

AND FRUIT

Thallic perchlorate in acid medium as a reagent for oxidimetric

determination of ascorbic acid in drugs and fruit has been studied 8. Thallic

perchlorate is used for determination of ascorbic acid either by direct titration in

acid medium (0.5N) or indirectly by addition of excess of the reagent and

iodometric determination of the excess. The method is applicable to fruit juices

and vitamin C tablets.

DETERMINATION OF TOTAL VITAMIN C IN FRUITS BY

CAPILLARY ZONE ELECTROPHORESIS MARCELLA CHIARIa, b,

MARINA NESIa, b, GIACOMO CARREAC AND PIER GIORGIO

RIGHETTIa, b

Determination of total vitamin c in fruits by capillary zone

electrophoresis marcella chiaria, b, marina nesia, b, giacomo carreac and pier

giorgio righettia, b
has been studied9. A simpje capillary zone electrophoretic

(CZE) method is described for the rapid determination of ascorbic acid

dehydroascorbic acid, the physiologically active forms of vitamin C, in fruits.

The electropheretic run was accomplished in 9 min on a coated capillary

column using 20mM phosphate buffer (pH 7.0). total ascorbic acid was

determined by first reducing the deydroascorbic acid to ascorbic acid by

treatement with DL-homocysteine. This reaction was complete in 15 min and

total ascorbic acid determination was performed immediately. The data obtained

by CZE were in good agreement with HPLC data.

QUANTITATIVE ESTIMATION OF VITAMIN C IN LOCAL FRUITS

Quantitative estimation of vitamin c in local fruits has been studied 10.

Four locally available and non-seasonal fruits namely: Adansonia digitata L.

(A). Ziziphus jujube L. (B). Dialium guineense W. (C). and the calyx Hibiscus

sabdariffa L. (D) were evaluated for their vitamin C (ascorbic acid) content

using an official methods (AOAC methods. 1980). The results obtained ranged

form 40-50mg vitamin C in 100g powdered dried fruits; the results showed the

vitamin C content sample C > B > D > A at 49.50, 44.60, 43.20 and

39.50mg/100g dried powdered samples respectively. Therefore, these fruits

when consumed will contribute to the supply the daily requirement the vitamin

by man.

MODIFIED METHOD FOR THE ESTIMATION OF ASCORBIC ACID

FROM PRESERVED JUICES AND SQUASHES

Modified method for the estimation of ascorbic acid from preserved

juices and squashes has been studied11. A displacement titration method for the

estimation of vitamin C from juices and squashes has been developed. Acetone

is used as complexing agent for bisulfate because formaldehyde is not suitable

as it reacts with N-bromosuccinimide and also combines with ascorbic acid. N-

Bromosuccinimide is used as the titrant, which preferentially oxidizes iodide to

iodine which in turn reacts with ascorbic acid. End-point is reached when bluish

colour persists for 30 seconds. The method is rapid and precise. The maximum

relative standard deviation is 2.4% in case of orange squash.

ASCORBIC ACID DETERMINATION IN COMMERCIAL FRUIT

JUICE SAMPLES BY CYCLIC VOLTAMMETRY

Ascorbic acid determination in commercial fruit juice samples by cyclic

voltammetry has been studied12. Ascorbic acid (vitamin C) is a water-soluble

vitamin which can be found in many biological systems and foodstuffs (fresh

vegetables and fruits, namely, citrus). Ascorbic acid plays an important role in

collagen biosynthesis, iron absorption, and immune response activation and is

involved in wound healing and osteogenesis. It also acts as a powerful

antioxidant which fights against free-radical induced diseases [1-5].

Nevertheless, an ascorbic acid excess can lead to gastric irritation, and the

metabolic product of vitamin C (oxalic acid) can cause renal problems [6]. In

some cases, excessive quantities of ascorbic acid may result in the inhibition of

natural processes occurring in food and can contribute to taste deterioration;

added to apple pulp (250 mg/kg), vitamin C inhibits oxidation processes

responsible for apple juice aroma [7]. Ascorbic acid is a labile substance, as it

easily degraded by enzymes and atmospheric oxygen. Its oxidation can be

accelerated by excessive heat, light, and heavy metal cations [1]. That is why

ascorbic acid content of foodstuffs and beverages represents a relevant indicator

of quality which has to be carefully monitored, regarding its variation during

manufacturing and storage.

THE COMPARISON OF VITAMIN C CONTENT IN COMERCIALLY

AVAILABLE SOURCES OF ORANGE JUICE USING HPLC

The comparison of vitamin c content in commercially available sources

of orange juice using HPLC has been studied 13.The present work utilized high

performance liquid chromatography (HPLC) method for determining vitamin C

content of commercially available orange juices by comparing juices in carton,

plastic jug, and frozen containers, as well as fresh orange. HPLC allows an

efficient and quick way to relatively quantify vitamin C with a low cost of

equipment and reagents. The results indicated higher daily percents of vitamin

C than those provided on the container’s label. The fresh sample yielded the

largest amount of vitamin C per serving, followed by the frozen sample, the

carton sample, and then the plastic jug sample.

In conclusion, it was shown that HPLC method can be used to determine

relative quantities of Vitamin C, but it is not an accurate method for determining

the absolute quantities in samples containing other organic acids. In this study,

the amount of ascorbic acid in orange juice is determined using HPLC without

separating ascorbic acid from the other organic acids present. The experiment
was able to rank containers with respect to vitamin C, the fresh sample have the

largest concentration, followed by the frozen sample, the carton sample, and the

plastic sample. The hypothesis was not correct. One cannot measure how much

ascorbic acid oxidizes in different containers because HPLC measures both

ascorbic and dehydroascorbic acids. For one to quantify only ascorbic acid

content within a sample, voltammetry and titration are two good methods to use.

Furthermore, voltammetry could be used because that method allows the

determination of one chemical compound within a mixture. Therefore, it cannot

be used to concruuently evaluate ascorbic and dehydroascorbic acids.

 EXPERIMENTAL METHOD

One way to determine the amount of vitamin C in food is to use a redox

titration. The redox reaction is better than an acid-base titration since there are

additional acids in a juice, but few of them interfere with the oxidation of

ascorbic acid by iodine.

Iodine is relatively insoluble, but this can be improved by complexing the

iodine with iodide to form triiodide:

I2 + I- <--> I3-

Triiodide oxidizes vitamin C to form dehydroascorbic acid:

C6H8O6 + I3- + H2O --> C6H6O6 + 3I- + 2H+

As long as vitamin C is present in the solution, the triiodide is converted

to the iodide ion very quickly. However, when the all the vitamin C is oxidized,

iodine and triiodide will be present, which react with starch to form a blue-black

complex. The blue-black color is the endpoint of the titration.

This titration procedure is appropriate for testing the amount of vitamin C

in vitamin C tablets, juices, and fresh, frozen, or packaged fruits and vegetables.
The titration can be performed using just iodine solution and not iodate, but the

iodate solution is more stable and gives a more accurate result.

PREPARING SOLUTIONS

1% Starch Indicator Solution

1. Add 0.50g soluble starch to 50 near-boiling distilled water.

2. Mix well and allow to cool before use. (doesn’t have to be 1%; 0.5% is
fine)

Iodine Solution

1. Dissolve 5.00g potassium iodide (KI) and 0.268g potassium iodate

(KIO3) in 200 ml of distilled water.

2. Add 30 ml of 3 M sulfuric acid.

3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final
volume of 500 ml of distilled water.

4. Mix the solution.

5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine
solution.
Vitamin C Standard Solution

1. Dissolve 0.250 g vitamin C ( ascorbic acid ) in 100 ml distilled water.

2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask
as your vitamin C standard solution.

Standardizing Solutions

1. Add 25.00 ml of vitamin C standard solution to a 125 ml Erlenmeyer

flask.

2. Add 10 drops of 1% starch solution.

3. Rinse your buret with a small volume of the iodine solution and then fill
it. Record the initial volume.

4. Titrate the solution until the endpoint is reached. This will be when you
see the first sign of blue color that persists after 20 seconds of swirling
the solution.

5. Record the final volume of iodine solution. The volume that was required
is the starting volume minus the final volume.

6. Repeat the titration at least twice more. The results should agree within
0.1 ml.
 You titrate samples exactly the same as you did your standard. Record the
initial and final volume of iodine solution required to produce the color change
at the endpoint.

Titrating Juice Samples

1. Add 25.00 ml of juice sample to a 125 ml Erlenmeyer flask.

2. Titrate until the endpoint is reached. (Add iodine solution until you get a
color that persists longer than 20 seconds.)

3. Repeat the titration until you have at least three measurement that agree
to within 0.1 ml.

Titrating Real Lemon

Real Lemon in nice to use because the maker lists vitamin C, so you can
compare your value with the packaged value.

1. Add 10.00 ml of Real Lemon into a 125 ml Erlenmeyer flask.

2. Titrate until you have at least three measurements that agree within 0.1
ml of iodine solution.

Samples:

 Fresh Fruit Juice – Strain the juice through a coffee filter or cheese cloth
to remove pulp and seeds, since they could get stuck in the glassware.
 Fruits & Vegetables – Blend a 100g sample with ~ 50 ml of distilled
water. Strain the mixture. Wash the filter with a few milliliters of distilled
water. Add distilled water to make a final solution of 100 ml in a
volumetric flask. Titrate these samples in the same way as the juice
sample described above.
REFERENCES

2. E. S. TEE, S. I YOUNG, S. K. HO* and S. SITI MIZURA Division of

Human Nutrition, Institute for Medical Research, Kuala Lumpur,

Malaysia.

3. William S. Smith, * Carlton Green, and Mungo Jerry Department of

Chemistry, stevens Institute of Technology, Castly Point on Hudson,

Hoboken, NJ 07030. Performed September 19, 2003; submitted

September 27, 2003.

Harris, D.C. Quantitative Chemical Analysis. 5 th ed.: W. H. Freeman &

Company, NewYork., 2000. Chapter 7.

4. Gweneth M. Chappell, M.Sc., And Audrey M. Hamilton, B.Sc.,

5. HENRY TAUBER and ISRAEL S. KLEINER

6. Chemistry and Materials Science Fresenius’ Journal of Analytical

Chemistry Volume 277, Number 5, 369-371.

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20133 Italy bFaculty of Pharmacy and Department of Biomedical

Sciences and Technologies, University of Milan, Via Celoria 2, Milan

 20133 Italy cIstituto di Chimica degli Ormoni, CNR, Via Mario Bianco 9,

Milan 20133 Italy Received 25 February 1993;

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as a Standard for Iodometric Titrations J. Chem. Educ., 1999, 76, 1421-

1422.

Gritsanapun, W.; Nilkamhank, S.; Paochom, A.; Suntornsuk, L.

Quantitation of vitamin C content in herbal juice using direct titration J.

Pharm. Biomed. Anal., 2002, 28, 849-855.

Kabasakalis, V; Moshatou, E.; Siopidou, D. Ascorbic acid content of

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