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1985, 57,555-558 555

(7) Headridge, J. B.; Riddington. J. M. Scanning Electron Microsc. 1981,
I I , 357-367.
(8) Flanagan, F. J. Geol. Sum. Prof. Pap. ( U . S . ) 1975, No. 8 4 0 ,
131-183.
(9) Gladney, E. S.;Burns, C. E.; Perrin, D. R.; Roehndts, I.: and Gills, T. E.
NBS Spec. Publ. ( U S . ) 1984, No. 260-08.
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(11) Wilson, A. D. Analyst(London) 1964, 89, 18.
(12) Benedetti-Pichler, A. A. I n "Physical Methods of Chemical Analysis";
Berl, W. M., Ed.; Academic Press: New York, 1956, Vol. 3, pp
183-21 7.
(13) Kratochvil, B.; Taylor, J. K. Anal. Chem. 1981, 53, 925A-938A.
(14) Ng, K. C.; Zerezghi, M.; Caruso, J. M. A n d . Chem. 1984, 56,
4 17-42 1.
(15) Korotev, R . L.; Lindstrom, D. J. Trans. Am. Nucl. SOC. 1982, 4 1 ,
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(16) Perlman. I.; Asaro, F. Archaeometry 1969, 7 7 , 21-52.
(17) Jacobs, F. S.; Filby, R. H. Anal. Chem. 1983, 55, 74-77.
(18) Filby, R. H.; Nguyen, S. N.; Grimm, C. A.; Markowski. G. M.; Ekambar-
am, V.; Tanaka, T.; Grossman, L. Proceedings of the 5th International
Conference on Nuclear Methods in Environmental Energy -. Research,
Mayaguez, PR, April 1984, in press.
(19) Hoffman, B. W.; Van Camerik, S. B. Anal. Chem. 1967, 39,
1198-1199.
(20) Davis, A. M.; Tanaka, T.; Grossman, L.; Lee, T.; Wasserburg, G. J.
Geochim. Cosmochim. Acta 1982, 4 6 , 1627-1651.
(21) Finkelman, R. B. Ph.D. Thesis, Department of Chemistry, University of
Maryland, 1980.
(22) Ingamells, C. 0.; Switzer, P. Talanta 1973, 2 0 , 547-568.
(23) Ondov, J. M.; Zoller, W. H.; Olmez, I.; Aras, N. K.; Gordon, G. E.:
Rancitelli, L. A.; Abel, K. H.; Filby, R. H.; Shah, K. R.; Ragaini, R. C.
Anal. Chem. 1975, 4 7 , 1102-1109.
(24) Fisher, G. L.; Prentice, B. A,; Silberrnan, D.: Ondov, J. M.; Biermann,
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(25) LyOn, W. S. I n Proceedings of the Nuclear Methods in Environmental
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RECEIVED for review August 6,1984. Accepted November 8,
1984. Supported in part by Electric Power Research Institute
under Contract FP 1259-1, under direction of M. McElroy
(MRI and WSU) and by the National Science Foundation
under Grant EAR-821-8154 and the National Aeronautical
and Space Administration under Grant NAG-9-54 (University
of Chicago).

Irreversible Reaction Kinetics of the Aerobic Oxidation of

Ascorbic Acid
David Emlyn Hughes
Norwich Eaton Pharmaceuticals, Inc.,' Norwich, New York 13815

Aerobic oxidation of ascorbic acid (AA) is studied at 25, 62,
75, and 86 'C. The AA is determined by 2,6-dichioroindo-
phenol titratlon and dehydroascorbic acid (DHA) is deter-
mined simultaneously by continuous-flow derivatlzatlon with
o -phenylenediamine using fluorescence detection. The
pseudo-first-order, reversible rate constants for the formation
of DHA and diketoguionic acid are discussed. The activation
energy for AA degradation and the irreversible pseudo-first-
order rate constant for AA and DHA loss are presented. A
degradation pathway from AA to products without the for-
mation of DHA is postulated.
Several mechanistic studies on the oxidation of AA have
been performed in the last 50 years. An excellent review of
the literature of aerobic and catalyzed oxidation is presented
by Mushran and Agrawal ( I ) . Definitive kinetic results may
have been unobtainable for several reasons: (1)the presence
of metal ions as impurities in laboratory water sufficient to
catalyze the reaction, (2) the absence of specific methods for
the determination of AA, and (3) the absence of precise and
sensitive methods for the determination of DHA.
Whereas under anaerobic conditions AA degrades to fur-
fural and carbon dioxide, the aerobic mechanistic path is
postulated to be oxidation to DHA followed by hydrolysis (2).
COOH

Ascorbic acid (AA) is an unsaturated lactone which is a 0 OH

strong reducing agent. It is converted to dehydroascorbic acid \/
(DHA) according to the reaction
I U
I
- FI
(2)
HO
I /
OH 0 0 CH2OH
dehydroascorbic acid
CHOH
I
CHOH AOH
0
+ 2Ht + 2e- (1) I
CHPH
oxalic acid
-I- other species
O=C C-H O=C, C
,H
-
0
'1
' 0 1 diketogulonlc acid
CHOH
I The degradation is apparently a function of at least the
I h20H concentrations of the metal ions present, the pH, and the
CH20H
available light ( 3 , 4 ) . The different kinetic routes apparently
ascorbic acid dehydroascorbic acid all produce DHA.
The reaction is apparently not reversible and is a function Hence, the oxidation of AA is assumed to occur by the initial
of at least the temperature, pH, and oxygen content of the formation of DHA followed by conversion of that species to
aqueous sample. The autooxidation is strongly catalyzed by diketogulonic acid (DKA) (5). The reactions are apparently
several metal ions, notably Cu(I1) and Fe(II1). pH dependent. In a more recent study, Blaug and Hajratwala
(6) determined the unreacted AA vs. time using the 2,6-di-
chloroindophenol volumetric determination. An apparent
' A Procter & Gamble Company. first-order rate of degradation was achieved at 67 "C and a
1.50/0
0003-2700/85/0357-0555$0 0 1985 American Chemical Society
556 ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
pH range of 3.5-7.2 a t an ionic strength of 0.4. The authors
postulated a mechanism in which undissociated AA, mono-
dissociated AA, and a complex formed by the two irreversibly
formed products.
Some studies ( 1 ) have been performed using the classical
2,4-dinitrophenylhydrazine derivative of DHA [Roe's (7)
method] which absorbs a t 500-550 nm after treatment with
85% sulfuric acid. The method does determine AA and DHA
but requires a 3-h incubation period. Okamura (8) used a
reduction of DHA with dithiothreitol followed by AA deter-
mination by the cup'-dipyridyl method which offers more
sensitivity than Roe's method. The classical 2,6-dichloro-
indophenol titration of AA has also been used and is quite
sensitive to metal ions but lacks specificity in the absence of
a suitable masking agent. Methods relying on ultraviolet
detection are hindered by the sensitivity of the AA ultraviolet
maximum to pH changes and the very low absorptivity of
DHA. High-pressure liquid chromatographic (HPLC)
methods (9) combine the above difficulties with kinetic
questions concerning the effects of residence of the species
in the mobile phase. Several spectrophotometric and titri-
metric procedures are available (10, 1 1 ) for the determination
of AA including the colorimetric procedure of Schmall mod-
ified to use p-nitroaniline (12) where sensitivity is not a
problem. In this paper, a procedure for simultaneously de-
termining AA and DHA will be presented. Kinetic data a t
several temperatures will be presented and a mechanism
proposed. This mechanism suggests that some traditional
vitamin C analyses may require careful interpretation.
EXPERIMENTAL SECTION
Materials. All reagents used in this study were of an ana-
lytically pure grade.
Methods. AA was determined by the 2,6-dichloroindophenol
volumetric method as modified by Hughes (13) for solutions
containing physically and chemically interfering species. The AA
standard solution, maintained at the cited temperatures, was
sampled periodically with a 2-mL pipet and titrated immediately.
Simultaneously, the DHA was monitored by continuous-flow
analysis. The sample line was combined with a 1mg/mL aqueous
solution of o-phenylenediamine and passed through a delay coil.
The resulting fluorescence due to the condensation product 3-
(1,2-dihydroxyethyl)furo[3,4-b]quinoxalin-l-one was then mea-
sured using a Technicon Autoanalyzer I1 Fluoronephelometer
equipped with a Corning No. 58 secondary filter and a Corning
No. 7-60 primary filter. Continuous flow was provided by a
Technicon Autoanalyzer proportioning pump. The general me-
thod has been used previously to determine the total vitamin C
content by prior oxidation with Norit (14), n-bromosuccinimide,
and other oxidizing agents. The availability of automated pro-
cedures has made this method more precise (15). However, since
some problems in the oxidation step have been reported in the
literature, the o-phenylenediamine procedure may not be satis-
factory for all determinations of AA. The procedure used here
has proven successful for the determination of DHA. This appears
to be the first time that this reaction scheme for the determination
of DHA alone has been reported in the literature. Five spikes
(100-1000 fig) of DHA to 1 mg/mL ascorbic acid solutions at a
pH of 2.6 and room temperature gave an average recovery of
101%. DHA standards were linear (RSD f 2 7 0 ) with a correlation
coefficient of 0.999 from 100 to 1000 Fg of DHA/mL.
KINETIC STUDY
Method of Analysis. An accurately weighed quantity of
AA (ca. 50 mg) was dissolved in water equilibrated to the water
bath temperature in a 100-mL volumetric flask. The contents
were immediately poured into a 125-mL Erlenmeyer flask in
contact with a water bath thermostated at the desired tem-
perature. Two-milliliter aliquots were removed periodically
for titration and a continuous-flow injection line was placed
in the flask to sample the dehydroascorbic acid. The initial
pH of the samples was measured. A bubbler was used to
maintain saturation of the solution with oxygen.
DISCUSSION
The implicit assumption of existing kinetic schemes is that
the mechanism of degradation of AA to DHA and DKA is
products products products
kI
DHA Re DKA
AA y T- (3)
characterized by comparatively large rate constants k , , k,,
kz, and k2; k , is assumed to be small; and the paths with rate
constants k , and k4 are not considered. As long as ascorbic
acid is the only species determined (as in previous studies),
the rate of disappearance of AA from reversible paths and
irreversible paths cannot be distinguished since ( k , + k 3 ) is
the apparent pseudo-first-order rate constant
-[AA] = k,[AA] + k,[AA] = (k, + k3)[AA] (4)
where [AA] = d[AA]/dt and the brackets indicate concen-
tration in bg/mL. With the addition of DHA degradation
concentration-time profiles, the role of the irreversible re-
actions of ascorbic acid becomes clearer. If the DHA ap-
pearance rate is lower than the ascorbic acid disappearance
rate, one or more of the kinetic routes with rate constants k,,
k4, or k , must be considered.significant. In the most
-
straightforward case, [AA] [DHA], that is, the absolute rate
of disappearance of AA approximates the rate of appearance
of DHA and rate constants k,, k,, k,, and k , are small com-
pared with the k l equilibration step.
T o test this simple case, the analytically correct form for
the concentrations according to Rodiguin (16) was employed,
i.e., for eq 3 with k,, k,, and k , = 0
PlPZ
Co = [AA] = Co'o'---- +
YlYZ
C1 = [DHA] =
Cz = [DKG] =
1
k1hZC0(O'-+ e-ylt + e-?*t (7)
Y1Y2 YI(Y1- YZ) Y d Y 2 - Yl)
where y1and y2 are the roots of the equation
with reversed signs. The rate constants were then determined
on a Hewlett-Packard 3357 with the experimental AA and
DHA concentrations as input. Using Rodiguin's nomenclature,
A. is AA, A , is DHA, A2 is DKA, k-, is PI, and k-? is p2, as
described in eq 3.
The kinetic data (Figures 1-3 and Table 11) were collected
a t 25 "C and pH 9.0, 62 "C and pH 2.6, 75 "C and pH 2.6,
and limited data at 86 "C and pH 2.6. The rate constants were
calculated assuming this simple mechanism. The results
appear on Table I. The activation energy for the reaction
was determined to be 12.0 kcal/mol at a pH 2.6 by the
standard Arrhenius plot of log k , vs. 1 / T , where T is the
absolute temperature. The activation energy is consistent with
the value of 12.2 kcal/mol, pH 3.52, given by Rogers (17).
 ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985 557

500 r

;JG/ML

250

0 30 60 90

TiME, MINUTES 0 30 60 90
Flgure 1. Kinetic data for the oxidation of ascorbic acid to dehydro-
TIME, MINUTES
ascorbic acid at 25 "C and pH 9.0: 0 represents ascorbic acid and
0 represents dehydroascorbic acid. Figure 2. Kinetic data for the oxidation of ascorbic acid to dehydro-
ascorbic acid at 62 "C and pH 2.6: 0 represents ascorbic acid and
0 represents dehydroascorbic acid.
Table I. Rate Constants for the Oxidation of Ascorbic
Acid"
temp, "C k,, min-' &, min-' k,, min-' &, min-I
62 8X 2X 1X 0
75 1.2 x 10-4 1 x 10-5 7 x 10-5 0
86 3 x 10-4 o 1 x 10-5 0
The exact solution for the mechanism eq 6 with k,, k4, and k , =
0 was utilized. k , and are the forward and reverse rate con-
stants for the AA to DHA conversion and k, and (3, are the forward
and reverse rate constants for the DHA to DKA conversion, re-
spectively. 250 1
Table 11. Concentration Profiles for Ascorbic Acid [AA]
and Dehydroascorbic Acid [DHA] at 86 O C and pH 2.6

time, min [AAI, d m L [DHAI, PLg/mL

10 479 66 _L-_-2-----J

20 440 137 0 30 60 90
30 403 200
40 369 250 TiME, MiNUTES
50 339 261
60 311 324 Figure 3. Kinetic data for the oxidation of ascorbic acid to dehydro-
70 285 ascorbic acid at 75 "C and pH 2.6: 0 represents ascorbic acid and
80 261 0 represents dehydroascorbic acid.
90 240
In the mathematically simplest form, R is not a function of
Although a reasonable set of rate constants was obtained time and eq 10 becomes
with the reversible reactions mechanism, the computer fit of [AA] + [DHA] = Rt f Ro (11)
DHA-time data was not satisfactory, since the predicted DHA
concentrations were higher than the experimental values. The where Ro is the initial sum of the concentrations of AA and
mechanism was therefore extended to include irreversible DHA. Hence, a graph of ([AA] + [DHA]) vs. time would be
paths to products. expected to be a straight line. For the AA data, the extrap-
If k l and k - , are much larger than the other rate constants olation formula

[AA] + [DHA] = 0 (8) [A] =

to the extent that AA and DHA are lost, then
[AA] + [DHA] = R
where R is some "global" rate of formation of products. Note
that this rate might itself contain a reversible reaction, e.g.,
DHA might be reversibly converted to DKA and then irre-
versibly to products, as in the accepted mechanism. Inte-
grating with respect to time, eq 9, becomes
[AA] + [DHA] = I R dt
was used since all previous AA degradation studies suggest
that the decomposition may be treated as a first-order reaction.
(9) The sum of [AA] plus [DHA] was then found and the resulting
function plotted vs. time of reaction. After an initial maximum
occurring in each case at typically 30-40 min, the ([AA] +
[DHA]) then decreases monotonically with time. Linear re-
gression of the data reveals that the relative standard deviation
for the 40- to 90-min data points is 1.4% for 25 "C, 0.81?G
for 62 "C, 0.41% for 75 "C, and 3.2% for 86 "C. The data
are seen to be linear with an average relative standard de-
(10) viation of 1.5%,which compares well with the expected rel-
558 ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985

T a b l e 111. R a t e C o n s t a n t s k, and k4 for the I r r e v e r s i b l e Loss of A s c o r b i c A c i d [ A A ] a n d D e h y d r o a s c o r b i c A c i d [DHA] by

Equation 3

temp, slope, intercept, A([AAl + [DHAl)/

"C PH unitless !.G/mL A t , fig mL-' min-' k,, min-' k,, min-'
25 9.0 -0.852 436 --0.785 1.8 x 10-3 1.5 x 10-3
62 2.6 -1.03 544 -0.201 3.7 x 10-4 3.8 x 10-4
75 2.6 -0.489 351 -0.772 2.2 x 10-3 1.1 x 10-3
86" 2.6 -0.444 300 -0.840 2.8 x 10-3 1.2 x 10-3
a Limited data a t this temperature.

ative standard deviation of f3-5% due to instrumental im-
precision.
If we now consider eq 3 with k 2 and k-, = 0, Le., two irre-
versible paths
-[AA] = + k3)[AA] k-,[DHA]
(121 - (13)
-[DHA] = (k-1 + k,)[DHA] - k,[AA]
combining eq 13 and 14
-([AA] = [DHA]) = k,[AA] + k,[DHA]
and hence
k4 [AA] + [DHA]
[AA] = - -[DHA] - (16)
k3 k3
A([AA] + [DHA])/At is a constant in the range t 2 40 min,
hence eq 16 takes the form
[AA] = m[DHA] +b
where m = -k,/k3 and b = A([AA] + [DHA])/k3At. A graph
of the AA concentration vs. DHA concentration allows the
calculation of irreversible rate constants k, and k,. Table I11
summarizes results of the calculations for k3 and k,. Rate
constants k , and k., represent total first-order loss of AA and
DHA. Rate constant k 3 represents the loss of AA by a
mechanism that does not have DHA as an intermediate.
Although the kinetic data presented here fit reasonably well
with the accepted AA-to-DHA-to-DKA mechanism (eq 6-9),
further analysis presented here suggests that an irreversible
path from AA to products appears to exist. The rate constant
k, appears to be large enough that it may be possible to
degrade measurable amounts of AA without any DHA being
formed. The concept that AA may react significantly with
several species is not new; in fact, Rogers (17) postulated
reaction with monodissociated AA, ascorbate ions and a
monodissociated AA-AA complex and presented rate con-
stants for the processes. The presence of an irreversible path
from ascorbic acid to products without intermediate DHA
formations requires some examination of accepted analytical
methodology. Vitamin C assays in which the AA is oxidized
titrimetrically, e.g., by the classical 2,6-dichloroindophenol (13)
procedure, are unaffected by the mechanistic details since
these assays only require that the AA present be oxidized to
some other species. Vitamin C assays ( 1 4 , 15) which rely on
the quantitative oxidation of ascorbic acid to dehydroascorbic
acid may be affected. Assays of this type require careful
evaluation since the AA to DHA concentration ratio in samples
is not under the control of the analyst. Hence, such a method
may perform well with AA standards and samples that contain
(14) a high percent of total vitamin C in the AA form. Assays of
predominately DHA samples may yield erroneous results.
With real samples, the analyst is not aware of the AA-to-DHA
(15) concentration ratio and is therefore unable to evaluate the
results correctly. The irreversible paths involving the com-
bined AA and DHA pool are of biological interest since only
AA and DHA show antiscorbutic activity. Hence, if there are
significant irreversible paths from AA and DHA to products,
the "total vitamin C", or sum of the AA and DHA, will de-
crease markedly with time. If the oxidation of AA may be
envisioned as solely the conversion to DHA, the total vitamin
C activity will be maintained.
(17) An irreversible path from AA to products without inter-
mediate DHA formation has a significant effect on the in-
terpretation of the kinetics of the oxidative mechanism and
analytical methods that rely on quantitative conversion.
LITERATURE CITED
(1) Mushran, S. P.; Agrawal, M. C. J. Sci. Ind. Res. 1977, 36 (6), 274.
(2) Connors. Kenneth A.; Amidon, Gordon L.; Kennon, Lloyd "Chemical
Stability of Pharmaceuticals"; Wiley: New York, 1979; p 138.
(3) Czuros. Z.; Petro, J. Acta Chim. Acad. Sci. Hung. 1955, 7 , 199.
(4) Rao, B. S. N. Radiat. Res. 1962, 77,683.
(5) Grochmalicka. J. Zesz. f r o b / . Postepow Nauk R o h . 1965, 53, 57;
Chem. Absrr. 1986, 64,5813f.
(6) Blaug, S. M.; Hajratwala, H. J. fharm. Sci. 1972, 61 (4), 556.
(7) Roe, J. H.; Kuether, C. A . J . Biol. Chem. 1943, 774,399.
(8) Okamura, M. Clin. Chim. Acta 1980, 703.259.
(9) Rose, R. C.; Narhwold, D. L. Anal. Biochern. 1981, 774,140.
(IO) Pandey, N. J. Anal. Chem. 1982, 54,793.
(11) Verma, K. K.; Glutai, A. K. Anal. Chem. 1980, 52. 2336.
(12) Weeks, C. A.; Deutsch. M. J. J. Assoc. Off. Anal. Chem. 1965, 48,
1245.
(13) Hughes, David Emlyn J. Pharm. Sci. 1983, 72,126.
(14) Deutsch, M. J.; Weeks, Cora E. J . Assoc. Off. Anal. Chem. 1965,
48, 1248.
(15) Roy, R. E.; Conetta, A.; Salpeter, J. J. ASSOC.Off. Anal. Chem.
1976, 59, 1244.
(16) Rodiguin, N. M.; Rodiguina, E. N. "Consecutive Chemical Reactions";
Van Nostrand: Princeton, NJ, 1964: p 42.
(17) Rogers, A. R.; Yacomeni, J. A. J. fharm. fharmacol. 1971, 23. 218.
RECEIVED for review July 30, 1984. Resubmitted November
5 , 1984. Accepted November 16, 1984.