Analysis of Vitamin C

by ~igh-pressureLiquid Chromatography
David E. Goodney
Willamette University, Salem, OR 97301

Primates, including human beings, cannot synthesize vitamin C (L-ascorbic acid) and must rely on their diets to
provide adequate amounts. T h e main sources of dietary vitamin C are fresh fruits and veeetahles. In addition, many
packaged foods are "fortified" k i t h additional vitamin C.
Vitamin C is also known to be easily oxidized to the less
potent dehydroascorbic acid. Thus vitamiu C remains a popular topic in the scientific literature and methods for the
determination of vitamin C in many types of food have been
reported (1-3). Vitamin C frequently appears in the popular
literature because of controversies over its effectiveness
against ailments ranging from the common cold to cancer.
The standard methods for analysis of ascorbic acid are the
2,6-dichloroindophenol titration based on the reducing ahility of ascorhic acid, or the o-phenylenediamine fluorescence
method which requires oxidation of ascorbic acid to dehydroascorbic acid ( I ) . I n recent sears several authors have
reported high-pressure liquid chromatographic (HPLC)
methods for ascorbic acid analysis (see references in review
l
experiments (4-7)
articles (2, 3)). I n this ~ o u r n a several
have been reported to determine the vitamin C in a variety of
samples, by a variety of methods except HPLC.
I report an HPLC method for the analysis of ascorbic acid
that can be used with a varietv of beveraee s a m ~ l e s .The
HPLC analysis is a modification of the procedure-reported
hv Dennisou e t al. (8)usine isocratic elution and UV absorbance detection.
method is rapid, reproducible, and
sensitive enough for ascorbic acid in the concentration range
investigated. HPLC with UV detection provides a convenient method of analysis of ascorhic acid in natural and
processed juices because the ascorbic acid is separated from
potential interferences, and it absorbs strongly in the UV
while most other constituents of juice do not. The ascorhic
acid is also separated from dehydroascorhic acid, but the
dehvdroascorbic acid does not absorb stronelv enoueh to be
The extent of oxidation of as&& acid in a
s a m ~ l eis determined hv reducine the dehvdroascorhic acid
and measuring the resulting "total ascorbic acid".

Typical Student Results

Sample
Orange Juice
(Frozen Concentrate)
Orange Juice
(Campus Foods)
Vitamin C Enriched
Beverage (Powdered)
Tomato Juice
(Canned)
Grapefruit Juice
(Fresh)
Grapefruit Juice
(Fresh)
Lemonade
(Frozen)

AsCOrblc Acid
wg/m~
25.6
. .. '
. 189

.
.

315

Total Ascorbic Acid

wglm~
39.9
3124
342

107
295

'

32.2

47.1

27.1

27.0

~ h k

Experimental
A Beckman model 342 HPLC with 4.6-mm X 25-cm Ultrasil-NHz
column was used for all the analyses. The mobile phase was a 4050
mixture of methanol: 0.25% KH2P04 huffer (KHzPOdHzO +
HIPO, adjusted to pH 3.5 before mixing). Detection was done by UV
absorption at 244 nm. At a flow rate of 1.25 mLImin., the retention
time for ascorbic acid was 7 min (Fig. 1).Sample size was 20 pL.
Standards for the HPLC analysis of ascorbic acid must be carefully prepared because of their low concentrations, and the possibility
of oxidation. The standard solution is prepared hy weighing 1g of
ascorbic acid to the nearest 0.1 mg and dissolving it in a 1-L volumetric flask using distilled water that has heen deoxygenated by
bubbling nitrogen through it. The standard should be prepared on
the day it is to be used and stored in the dark. Dilutions of the
standard in the 25-250 pg1mL range are used to generate a calibration curve of peak height versus concentration for quantitative analysis. Approximately 3 mL of each standard are filtered through a
0.45-pm filter prior to injection into the chromatograph.
Quantitative analysis of ascorbic acid can be done on samples of
fresh,canned, frozen, or powdered beverage. Students bring to class
their own sample of beverage containingvitamin C and, if necessary,

Time (M,",

Chmmatograms of solutions containing vitamin C. (A) Standard solution containing 101 pglmL of ascorbic acid. (B) Diluted sample of vitamin C enriched
beverage. (C) Beverage sample after reduction. Note the different absorbance
scale on A compared with Band 0.

prepare it according to package instructions. Exposure to light is

minimized. The beverage is diluted by pipeting 5 mL into a 50-mL
volumetric flask and filling to the mark with deoxvrcenated water.
Approximately 3 mL of t<e diluted samole are filtered through a
0.45-pm filter.
To determine the extent of$sidation of the ascorhic in the sample, 5 mL of the undiluted b
w are pipeted into a small beaker,
va~lablefrom Sigma Chemicals;
20 mL of a 0.8% homocyste
keep refrigerated) solution asded, and the pH adjusted to 7 with a
45% K2HP04huffer (pH 7.0). A minimum of huffer and pH paper
are used for the adjustrnent.'The homocystaine will quantitatively
reduce dehydroascorbic acid to ascorbic acid. After 15 min, transfer
the solution to a 50-mL volumetric flask and dilute to volume with
deoxygenated water. Approximately 3 mL of the solution are filtered through a 0.45-pm filter. .,
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Volume 64

~umber2

Februarv 1987

187

Chromatograms are recorded for replicate injections of the standards, sample, and reduced sample (see figure). Approximately 10
min are required for each chromatogram. Several new peaks appear
in the chromatogram of the reduced sample. These new peaks are
artifactsof the reduction and have not been identified. The height of
the ascorbic acid peak is used for quantitative calculations.
Discussion

A calibration curve is constructed hv

plotting peak h e i ~ h t
..
versus concentration from the standard chr&atograms.
The concentrations of ascorbic acid in the sample and homocysteine treated solutions are then taken from the calibration curve. Multiplication by the dilution factor of 10 yields
the
of ascorbic acid and "total ascorhic acid"
~ - concentration
~ in the beverage. The total ascorbicacid is the sum of ascorhic
acid and dehvdroascorbic orieinallv
" in the beverage.
Student analyses of heverages yields results Gee table)
that are consistent with the range of values (37-600 d m L )
reported by several authors (8,10-11) for the axorb& acid
content of beverages. Students report concentrations of
ascorbic acid that range from close to zero > 100 pglmL,
depending on the sample chosen, its age, and presence of
~~~~~~~~~

~~~

added vitamin C. The value of 3124 pg/mL for the total

ascorbic acid in orange juice from campus foods appears to
have been juice fortified with vitamin C, most of which had
oxidized. As expected the "total ascorbic acid" is greater
than or equal to the ascorhic acid. The larger the difference
between them, the greater the extent of oxidation of ascorhic
acid in the beverage. A recent paper by Hughes (9) strongly
asserts that the oxidation of ascorhic acid is complicated and
that products other than dehydroascorhic acid may be irreversibly produced. Thus the amount of vitamin C originally
present may have been even greater than the total ascorhic
acid determined by this method.
Llterature Cited
1. O//i<id Merhnda of AnoI.y&. 14th ed., Assodation of Official Analytical Chemists:
Arlington, VA, 19M pp 84G346.
2. Fult.. A. K.: Yeranrim,
K.G. A n a l Clwm. 1983.55.164R.
3. Yernnrian, J.
Slnman. K. G.;
K. A n a l Chem. 1985.57.273R.
4.
F.J. Chem.Educ. 1972.49,738.
5 . Marsh, D.
L.;
J.Chem.Educ.
6. Railey, D.N.J. Chem Educ. 1974.51.488.

J.A.:Siomsn,
A.:
Fo1tz.A.
Fulkrod.d.
G.;Jacobi,D. Veening,H.
1913.50.626.
7. Simta. G.R.: Maelnnir, W. K.:Raamussen.P.W. J . Chem.Edue. 1919.S6.421.
8. Dennir0n.D. B.: Braw1ey.T. G.;Hunter, G. L. J.Agric.FoodChsm. 1981,29,927.
9. H U ~ E,
~ n~n aSi them.
, ~ 1986,5i,
.
555.
10. Rrme,R. C.; Nahrwaid. D. L.Anal. Riocham. 1981,111,140.
11. Camevale. J. Food Technol.Ausrm1. 1980.32.302.