[14] A n a l y s i s o f T o t a l P h e n o l s a n d O t h e r O x i d a t i o n
Substrates and Antioxidants by Means of
Folin-Ciocalteu Reagent
By VERNON L. SINGLETON, R U D O L F O R T H O F E R ,
and ROSA M. LAMUELA-RAVENT6S
Introduction
Phenols occurring in nature and the environment are of interest from
many viewpoints (antioxidants, astringency, bitterness, browning reactions,
color, oxidation substrates, protein constituents, etc.). In addition to simple
benzene derivatives, the group includes hydroxycinnamates, tocopherols,
and flavonoids in plants and foods from them, tyrosine and DOPA deriva-
tives in animal products, and additives such as propyl gallate in foods.
Estimation of these compounds as a group can be very informative, but
obviously not simple to accomplish. Isolative methods such as high-perfor-
mance liquid chromatography (HPLC) are difficult to apply to such a
diverse group having, furthermore, many individual compounds within each
subgroup. Interpretation of such results is even more difficult.
Phenols are responsible for the majority of the oxygen capacity in most
plant-derived products, such as wine. With a few exceptions such as caro-
tene, the antioxidants in foods are phenols. Among those added to prevent
oxidative rancidity in fats are the monophenols (benzene derivatives with
a single free hydroxyl group) 2,6-di-tert-butyl-4-hydroxytoluene (BHT) and
its monobutylated anisole analog (BHA). tert-Butyl substituents function
mainly to increase the lipid solubility. In aqueous solution the parent mono-
phenols and others can also function as antioxidants. Therefore, it is impor-
tant that total phenol assays include monophenols as well as more easily
oxidized polyphenols.
An antioxidant effect can be from competitive consumption of the
oxidant, thus sparing the target molecules being protected, and from
quenching the chain reaction propagating free radical oxidation. Antioxi-
dants become oxidized as they interfere with the oxidation of lipids and
other species. Paradoxically, because of coproduction of hydrogen peroxide
as an antioxidant phenol or ascobic acid reacts with oxygen, coupled oxida-
tion can occur of substrates (ethanol, for example) that would not react
readily with oxygen alone. 1
2 j. A. Rossi, Jr. and V. L. Singleton, Am. J. Enol. Vitic. 17, 231 (1966).
3 V. L. Singleton, Am. J. Enol. Vitic. 38, 69 (1987).
4 j. j. L. Cillers and V. L. Singleton, J. Agric. Food Chem. 37, 890 (1989).
5 0 . Folin and V. Ciocalteu, J. BioL Chem. 73, 627 (1927).
6 V. L. Singleton and P. Esau, Adv. Food Res., Suppl. 1, 1 (1969).
154 POLYPHENOLS AND FLAVONOIDS [ l 41
Reagent
Folin and Denis s first proposed a heteropoly phosphotungstate-
molybdate reagent to react with tyrosine to give a blue color proportionate
7 A. Scalbert, in "Plant Polyphenols" (R. W. Hemingway and P. E. Laks, eds.), p. 259. Plenum
Press, New York, 1992.
s O. Folin and W. Denis, J. Biol. Chem. 12, 239 (1912).
[ 141 FOLIN-CIOCALTEUREAGENT 155
Procedure
The manual method 9 calls for 1.00 ml of sample, blank, or standard in
water (or dilute aqueous solution) added to at least 60 ml of distilled water
in a 100-ml volumetric flask. Add FCR (5.0 ml) and mix. After 1 min and
before 8 min, add 15 ml of 20% sodium carbonate solution, adjust the
volume to 100.0 ml, and read the color generated after about 2 hr at about
23 ° at 760 nm in a 1-cm cuvette. Provided appropriate standards and blanks
are employed, considerable variation in these conditions may be permissi-
25.5" C
0.:32
0.22 / 40°C
i I, , I I I I I
2 4 6 8
HOURS
FIG. 1. Absorbance development from FCR and FDR with time at two temperatures and
with 2.0 g/100 ml (solid lines) or 3.0 g/ml (dashed lines) sodium carbonate. Reproduced with
permission from V. L. Singleton and J. A. Rossi, Jr., A m . J. EnoL Vitic. 16, 144 (1965).
~2M. Celeste, C. Tomas, A. Cladera, J. M. Estela, and V. Cerda, Anal. Chim. Acta 269,
21 (1992).
158 POLYPHENOLSAND FLAVONOIDS [ 141
The standard solution (500 mg GAE/liter) may be used as the top level
in a series of standard dilutions, but its blue pigment production will be
outside the absorbance range for satisfactory spectrophotometry in stan-
dard equipment. If standard and unknown samples prove to be too high,
dilution of the blue color can indicate the proper phenol level of the samples,
but they should also be diluted and reanalyzed. The measurable color yield
should be linear or nearly so up to about 300 mg GAE/liter in the sample
by the procedure described. Sample content is expressed most simply by
direct computation from a plot prepared from standard dilutions with the
same portion volumes as the samples rather than on the basis of the final
reaction volume. The minimum detectable amount is of the order of 3 mg
GAE/liter, especially if the sensitivity is extended by such techniques as
cuvettes with longer light paths. Remember that oxidation, from air or
otherwise, can alter stored samples, especially at low content.
0.6 I i I I I
WC
0.5
~0.4 -
°0.3 . ~ . . -
0.2
0. I i I I I I
500 600 -tOO 800
WAVELENGTH, nrn
FIo. 2. Absorption spectra produced by wine (W) and gallic acid (G) with Folin-Ciocalteu
(C) and Folin-Denis (D) reagents. Reproduced with permission from V. L. Singleton and
J. A. Rossi, Jr., A m . J. EnoL Vitic. 16, 144 (1965).
a Adapted with permission from V. L. Singleton, Adv. Chem. Ser. 134, 184 (1974).
[ 141 FOLIN-CIOCALTEU REAGENT 163
Other studies, z7'18 allowing for changed conditions, including the use of
FDR, have generally agreed very well in relative terms, even if not in
absolute molar absorptivity, and have added new compounds. Under stan-
dardized conditions of the FC analysis, phenol itself gave 12,700 molar
absorbance. 16 Most other biologically likely monophenols tested gave simi-
lar or slightly higher (to 15,900) extinctions. Electron-attracting groups
such as chloro, carbonyl, or nitro gave progressively less extinctions (4-
chlorophenol, 12,100; salicylic acid, 8000; and picric acid, 0). Phloroglucinol
and nearly all other meta-polyphenols reacted as monophenols, as did para-
hydroquinols. Catechols and pyrogallol derivatives with free hydroxyls gave
twice the color of monophenols in agreement with their ortho-quinone
possibilities. Resorcinol behaved as a diphenol, but its derivatives generally
tested as monophenols.
Flavonoids such as catechin closely approximated the sum of the color
expected from the phloroglucinol A ring plus the reaction possibilities of
their B ring (i.e., one plus two in this case). Flavonols such as quercetin,
but not their 3-glycosides, gave more color than predicted, probably from
participation of the enolic C ring. This idea is reinforced by the behavior
of flavone and flavanones, which lack phenolic groups (Table I).
Generally, methoxyl substitution removed the reactivity of that phenolic
group, but in some instances, particularly sinapic acid, there was indication
of partial removal of a methyl group under assay conditions to generate
additional free phenolic hydroxyls and additional blue pigment. Despite
the alkaline conditions, ester and lactone formations involving phenolic
hydroxyls appear to remain intact and inactive during the assay, at least at
room temperature.
Reinforced with recovery from mixtures of phenols, ~6 these data show
that a good first approximation of the contribution of a given weight of a
specific compound to total color by the FC assay can be made by calculation
taking into account the number of predicted reacting groups and the molec-
ular weight compared to the gallic acid standard. Even better estimate can
be made if the color yield of the specific compound in question is compared
experimentally to gallic acid under the specific reaction conditions in use.
It is unreasonable to expect that the total UV-HPLC peak area will correlate
with total phenol by FC unless such calculations have been made. A high
UV absorber may be a low contributor to the phenolic total and vice versa.
17T. Swain and J. L. Goldstein, in "Methods in Polyphenol Chemistry" (J. B. Pridham, ed.),
p. 131. Macmillan, New York, 1964.
is M. Haug, B. Enssle, M. H. Goldbach, and K. Gierschner, Ind. Obst. Gemueseverwert. 69,
567 (1984).
164 POLYPHENOLSAND FLAVONOIDS [ 14]
Interferences
The inclusiveness of the oxidation due to the FCR makes it unsurprising
that the analytical result can include "interfering" substances in many
crude, natural samples. If the possible interfering substances and their likely
concentrations are known, efforts to limit the FCR assay to phenols can
often be successful. To a degree the assay should be considered a measure
of oxidizable substrates, not just phenols. In any case, results can be very
useful, if interpreted properly.
Interferences can be of three types: inhibitory, additive, and enhancing
or augmenting. Conceivably, inhibition could be from oxidants competing
with the FCR, but such a reaction in samples should have been completed
in advance. Air oxidation after the sample is made alkaline can certainly
decrease phenols oxidizable by FCR. This is a reason why FCR addition
ahead of alkali has been emphasized. If this is done, exposure is limited
and is the same for samples and standards. Efforts to sparge and blanket
with nitrogen have shown no significant effect. Inclusion of solvents other
than water in the samples has sometimes inhibited color formation, but in
practical usage the effect has been small or avoidable by solvent change
or correctable by matching standards and blanks with the samples.
Additive effects are to be expected if unanticipated phenols or enols
(e.g., additives or microbial metabolites) are present, as will also be the
case with nonphenol FCR reactants. Aromatic amines as well as aminophe-
nols are included in assays for total phenol (Table I). Tryptophan and other
indoles react quantitatively with FCR, as do some purines. This has been
known for a long time. 5's Guanine (but not guanosine), xanthine, and uric
acid reacted to give molar color yields with FCR equivalent to monophe-
nols. 19,2° Adenine and other purines and the common pyrimidines gave
little color formation (about 1/50th as much). Alkaloids such as caffeine
do not appear to have been tested. In wines and many other samples,
insufficient of these compounds is present to contribute importantly in the
assay for total phenols.
The reaction of proteins with FCR sometimes has been considered an
interference, but this is somewhat unfair as most of the reaction is from
tyrosine and tryptophan content. Further confusion has arisen from the fact
that the Lowry method of protein determination 2t,22 uses FCR. However, in
this method alkali is added and incubated with copper ions well before
TABLE II
APPROXIMATE CORRECTIONS FOR INVERT SUGAR CONTENT FOR FC DETERMINATION
OF TOTAL PHENOLSa'b
A B
a Adapted with permission from K. Slinkard and V. L. Singleton, Am. J. Enol. Vitic. 28,
49 (1977).
b FC conditions A = 25 °, 120 rain; B = 55 °, 5 min. For instance, for a sample with 5.0%
inverted sucrose under condition A, an apparent total phenol of 1000 mg GAE/liter
should be corrected by 6% to give 940 mg GAE/liter.
20j. Bloin, L. Llorca, F. R. Montreau, and J. H. Dufour, Connaiss. Vigne Vin. 4, 403 (1972).
27 F. W. Mtlller-Augustenberg and H. Kretzdorn, Dtsch. Wein-Ztg. 91, 314 (1955).
[ 141 FOLIN-CIOCALTEUREAGENT 167
2sT. C. Somers and G. Ziemelis,J. Sci. Food Agric. 31, 600 (1979).
29G. Schlottenand M. Kacprowski,Wein-Wiss. 48, 33 (1993).
30S. D'Agostino, Vignevini 13(10), 17 (1986).
31M. Moutounet, Connaiss. Vigne Vin. 15, 287 (1981).
168 POLYPHENOLSAND FLAVONOIDS [14]
4~ I I I I
.~ B~
hi
2C
)-
u)
I0
I I I
0 I0 2O 50 4O 5O
Mg/L PHLOROGLUCINOL
F[G. 3. Total phenol by Folin-Ciocalteu assay of phloroglucinol alone (A) and in the
presence of 23 rag/liter of freshly added SO2 (B).
[ 141 FOLIN-CIOCALTEU REAGENT 169
601 I - - F ...... ] I
13
d
/
/
!.9
I
i
I
>- /
< /
20 t
__1
o /
z /
:]: /
a. /
10 il
/
1 /
/
/
k¢ I I I I I
0 10 20 50 40 50
Mg/L GALLIC ACID
FIG, 4. Total phenol by Fo|in-Ciocalteu assay of gallic acid alone (A), with freshly added
SO2 at 24.2 mg/liter (B), 25.6 rag/liter SO2 + 35 rag/liter acetaldehyde (C), and 25.3 mg/liter
SO2 + 79 rag/liter acetaldehyde (D).
E
>_-
<I
or)
or)
• k
-o F
20C oE
b.I ,D
, oC
O. "~'•'~'~-. • __ o. ,,
"B
, , ,, • A
7 I , I. . . . . I
W
0 I000 20O0 5000
ACETALDEHYDE, mg/liler
FIo. 5. Total phenol assay by Folin-Ciocalteu of a Riesling white wine alone (W) and
with various levels of acetaldehyde following the addition of SOz in nag/liter of A = 25,
B = 50, C = 100, D = 150, E = 200, and F = 300.
red wines at considerably higher total phenol levels, suggested total contri-
butions of about 100 mg GAE/liter in typical light white wines without
fermentable sugar. Such wines contain interfering substances in small but
contributory amounts (bound sulfites, some purines, some dehydroascorbic
acid, etc.) accounting for most, if not all, of the excess of the total phenol
of FC over the sum of the readily identified individual phenols. Of course
the comparison method may be at fault. Good relative values are possible
routinely with FC and, with the consideration of potentially interfering
substances and the levels present, more certainty is achieved.
Differential M e t h o d s a n d Variations
The total phenol by FC can be compared with values by other analyses
on the same samples for subgroups such as tannins, flavonoids, anthocya-
nins, or phenolic acids. Among such other analyses are 520 nm absorbance
for anthocyanins, leucoanthocyanidin conversion to cyanidin, and vaniUin
colorimetry for certain flavonoids. Especially for samples with high phenol
content such as red wines, such comparisons have been useful, for example,
[ 141 FOLIN-CIOCALTEU REAGENT 171
32E. Carruba, S. D'Agostino, B. Pastena, C. Alagna, and G. Torina, Riv. Vitic. Enol. 35,
47 (1982).
33E. LaNone and D. Antonacci,Riv. Vitic. Enol. 38, 367 (1985).
34S. Cliffe, M. S. Fawer, G. Maier, K. Takata, and G. Ritter, J. Agric. Food Chem. 42,
1824 (1994).
35A. C. Jennings,A n a l Biochem. 118, 396 (1981).
36T. E. Kramlingand V. L. Singleton,Am. J. Enol. Vitic. 20, 86 (1969).
172 POLYPHENOLS AND FLAVONOIDS [141
tion will be incomplete. If too high, the active sites (six and eight on
catechin) can be excessively converted to methylol (soluble) derivatives
and cross-linking decreased. Furthermore, the precipitate is soluble in 95%
ethanol and appreciably in other aqueous solvents, including those from
formaldehyde.
Formaldehyde at 40 mg per sample is about 15-fold maximum molar
equivalent of the reactive phenols in higher samples in the indicated range
and gave slightly more complete precipitation than did 120 mg per sample. 36
The formaldehyde solution was freshly prepared by dilution of 2.1 ml of
36% formalin to 100 ml. Note that the formaldehyde solution was listed
erroneously as much too concentrated in some previous citations. 37'38
After 24 hr, the reaction mixture is centrifuged or the supernatant
decanted, and, if not perfectly clear, filtered through a 0.45-/~m membrane
filter. The residual nonflavonoid content is determined as usual by FC.
Remember to compensate for the dilution compared to the original sample
by either doubling the result or analyzing double the aliquot. Separately
determine the total phenol by FC on the original, untreated sample. The
total minus the nonflavonoid phenol (GAE or other matching units) equals
the flavonoid content of the sample. The coefficient of variability of re-
peated nonflavonoid was about 3% and flavonoid values, because they
involved two assays, were about double.
Obviously, if flavonoids are precipitated incompletely, the apparent
nonflavonoids will be too high and if nonflavonoids are coprecipitated,
too low. Less obviously, because sulfite augmentation would be swamped
automatically by the formaldehyde, nonflavonoids by this procedure will
be only subject to addition by sulfite, but unless the original total phenol
was corrected to prevent any augmentation, the apparent flavonoid content
could be too high. For a very low flavonoid content (<40 mg/liter), FC
overestimated flavonoid content and had high variability, making it difficult
to ascertain the completeness of removal of flavonoids by hyperoxidation
of juices. 39 Also note that flavonoids with 4-carbonyl groups are deactivated
and resistant to reaction with formaldehyde.
With some samples low in flavonoids, only a haze results from formalde-
hyde treatment. Conversely, anthocyanin glycosides, although flavonoids,
are too soluble to precipitate completely unless sufficient condensed tannin
or another less soluble phenol is available for cross-linking. Because phloro-
glucinol is precipitated completely by acidic formaldehyde, addition can
solve both problems. Faint pink may still remain in the strongly acidic
solution even after phloroglucinol addition, but by spectral analysis this
represents only a few milligrams of flavylium ions and an insignificant
contribution to FC totals.
In general, phloroglucinol has not been added unless necessary for the
two situations mentioned. When it is added, the procedure 37"38is modified
as follows: 10.0 ml of sample, 2.0 ml of concentrated HCI, and 5.0 ml of 8
mg/ml formaldehyde for low flavonoid samples and 12 mg/ml for high ones
are well mixed and allowed to stand at room temperature for 2-3 hr. Then
3.0 ml of 10 mg/ml phloroglucinol is mixed into high anthocyanin samples
or 1.0 ml of the same plus 2.0 ml of water into those with low flavonoids.
After a total reaction time of about 24 hr, the FC analysis is completed
as before.
As is the case with most such reactions, the formation of a high amount
of precipitate can adsorb phenols that were otherwise soluble. Because of
the high acidity, it may be necessary to raise the concentration of the sodium
carbonate used in the FC analysis, which should be checked under the
specific conditions employed.
This flavonid-nonflavonoid separation is not as precise as one would
like. A few nonflavonoids precipitate slightly with formaldehyde, ~6 fisetin
(resorcinol A ring) did not, and flavonoid glycosides alone are incompletely
precipitated. However, the analysis has led to important findings. Nonfla-
vonoid phenols in grapes and wines, mostly hydroxycinnamates, are rela-
tively similar regardless of processing, whereas the flavonoid content rises
greatly as pomace extraction O c c u r s . 36 However, in apple juices a similarly
wide range of total phenol content can be found, but the nonflavonoid
content maintains a relatively constant fraction of the total. 4° Tannins ex-
tractable from oak barrels are hydrolyzable (not flavonoid) whereas those
present from the grape are condensed (flavonoid) tannins. Because grape
phenols other than flavonoid are low and relatively constant, oak barrel
extraction during wine (or spirit) aging or tannic acid addition can be
quantitated. 37 Scalbert et aL 41 found good agreement with these results for
a series of nonflavonoid tannins from various woods, but found that with
added phloroglucinol these tannins and small, less soluble phenols were
entrained appreciably.
Folin-Ciocalteu analysis has been applied before and after treatment
with lead acetate, gelatin, hide powder, polyvinylpyrrolidone (PVP), poly-
amide, cinchonine, methylcellulose, sodium chloride, and ethyl acetate ex-
traction. The first three can be passed over because lead acetate precipita-
47 C. Pompei, C. Peri, G. Montedoro, E. Miniati, and N. Pasquini, Ann. Technol. Agric. 20,
21 (1971).
48 S. Mitjavila, M. Schiavon, and R. Derache, Ann. TechnoL Agric. 20, 335 (1971).
49 C. Peri and C. Pompei, Phytochemistry 10, 2187 (1971).
50 A. Brugirard and J. Tavernier, Ann. Technol. Agric. 1, 311 (1952).
51 C. Peri and C. Pompei, Am. J. EnoL Vitic. 22, 55 (1971).
176 POLYPHENOLS AND FLAVONOIDS I14]
liter), the proportion of phenol precipitated by cinchonine increased and
was, respectively, 32.1, 45.2, and 52.8% of the total FC phenol. 43-46 Con-
versely, the proportion of the FC total phenol extractable by ethyl acetate
decreased and was, respectively, 36.1, 24.7, and 14.6% of the original total.
If one assumes no overlap between those phenols extracted by ethyl acetate
(nonpolar, dimers and smaller) and those precipitated by cinchonine (tan-
nins equivalent to trimers and larger), there was 30-33% of the total phenol
attributable to nontannin, nonextractable phenols, i.e., polar compounds
such as caftaric acid (caffeoyltartaric acid), and glycosides.
Montedoro and Fantozzi, s2 with additions of highly methylated methyl-
cellulose at double or more the tannin content, at pH 3-5, and with 100
g/liter of ammonium sulfate, did not precipitate hydroxy-substituted benzo-
ates, cinnamates, simple phenols, flavonols, catechins, or phenolic glyco-
sides, whereas cinchonine did precipitate portions of the last three. All
three methods did precipitate gallotannic acid; methylcellulose more than
PVP and slightly less than cinchonine. With model compounds5e and a wide
series of beverages, 53 recovery of added tannic acid was good and repeated
analyses had coefficients of variation of 1.3-13.1%. Formaldehyde precipita-
tion on the supernatant after methylcellulose adsorption gives nontannin
nonflavonoids and nontannin flavonoids by difference.
As just discussed, the three most widely applied methods to fractionate
the large, FC-active phenols are precipitation with polyvinylpyrrolidone,48
cinchonine sulfate,49 or methylcellulose.52Burkhardt 54 found on a must and
red wines prepared from it with increasing degrees of pomace extraction
that the three methods were equally suitable for laboratory analysis. For
the three samples with the lowest phenol (530-780 mg CtE/liter), removal
by cinchonine, PVP, and methylcellulose averaged, respectively, 1.3, 11.7,
and 34.0% of the total FC phenol. For the three highest wines (2310-3280
mg CtE/liter), respective average removals were 48.7, 52.7, and 44.7%.
From these data it appears that PVP had slightly greater affinity for the
larger phenol molecules and methylcellulose a greater affinity in the dilute
solution among mostly nontannin phenols. Unfortunately, formaldehyde
precipitation was not included in these tests.
By a statistical study55 of the analytical separation of total phenols by
sodium chloride precipitation, formaldehyde precipitation, and comparison
with other analyses and ratios, 93-100% of a group of red, pink, and wines
made from a mixture of white and red grapes were classified correctly into
[15] S i z e S e p a r a t i o n o f C o n d e n s e d T a n n i n s b y
Normal-Phase High-Performance Liquid Chromatography
B y V E R O N I Q U E CHEYNIER, J E A N - M A R C SOUQUET, E R W A N LE Roux,
SYLVAIN G U Y O T , a n d JACQUES R1GAUD
Introduction
Condensed tannins, also called proanthocyanidins because they release
anthocyanins when heated in acidic conditions, are ubiquitous plant compo-
nents, consisting of chains of flavan-3-ol units (Fig. 1). Several classes can
be distinguished on the basis of the hydroxylation pattern of the constitutive
units. Among them, procyanidins, composed of (epi)catechin units (Fig. 1,
R3 = H), and prodelphinidins, deriving from (epi)gallocatechin (Fig. 1,
R3 = OH), are particularly widespread.
Within each class, monomeric units may be linked by C-4-C-6 and/or
C-4-C-8 bonds (B type) or doubly linked, with an additional ether linkage
(A type) and eventually substituted (e.g., glycosylated, galloylated). The
structures of numerous oligomers have been elucidated) However, the
lower molecular weight proanthocyanidins are usually present in relatively
low concentrations compared to polymers, a Besides, the degree of polymer-
ization (DP) may vary greatly, as proanthocyanidins have been described
up to 20,000 in molecular weight. 3
Tannin properties, including radical scavenging effects and protein-
binding ability, depend largely on their structure and particularly on the
number of constitutive units (DP). Therefore, several methods have been
1 L. J. Porter, in "The Flavonoids: Advances in Research since 1984" (J. B. Harborne, ed.),
p. 23. Chapman and Hall, London, 1994.
2 Z. Czochanska, L. Y. Foo, R. H. Newman, and J. L. Porter, J. Chem. Soc. Perkin Trans I
2278 (1980).
3 E. Haslam and T. H. Lilley, Crit. Rev. Food Sci. Nutr. 27, 1 (1988).