COLORIMETRY OF TOTAL PHENOLICS W I T H
PHOSPHOMOLYBDIC-PHOSPHOTUNGSTIC
A C I D REAGENTS
V. L. SINGLETON and JOSEPH A. ROSSi, JR.J
The official analytical method of the
Association of Official Agricultural Chem-
ists for "tannin" in wines and spirits (7/ is
based upon fh,e phenol reagent of Folin
and Denis (3). Variations of this method
are widely used fo determine phenolic sub-
stances in many foods and other materials.
If is considered the method of choice for
estimating fofai phenol content in com-
plex plant products (20) and has been
adopted for this purpose in our studies
of the nature and imporf.ance of phenolic
substances of grapes and wines as related
fo flavor, storage changes, etc. (19).
Dir,ecf application of the officiai
A.O.A.C. method or descriptions in other
reference works (e.g. I, 7, 8, 18) often
leads fo difficulties such as the formation
of a troublesome precipitate, deviation
from the Be,or-Lambert law (with higher
phenol content), and appreciabe varia-
tion between different analyses un!ess
conditions are duplicated exactly. Since
the color yield may vary for different
substances the values obtained on complex
natural mixfupes are somewhat empirical.
Considering this empiricism and the dif-
ficulties mentioned, if is perhaps not sur-
prising fhaf the specific details of the
methods of this type in use in various
laboratories differ considerably. These varl-
etions inciud,e maior differences in the
•nature and concentration of the reagents
used, the time sequence of reaction and
color measurement, and the wavelength
af which the absorbanoe is determined.
For- example, the intensify, of the blue
cobr produced in the reaction has been
measured by various authors af 520, 660,
' Respectively Associate Enologisf and (6raduafe
Student, Department of Viticulture and Enology,
University of California, Davis.
144
725, and 760m~, and af other wavelengths
in this wide range.
While if appears fhaf the values ob-
tained by each analyst with his particular
method can give informative relative values
within a given set of analyses, the compar-
abiiify of results obtained under different
circumsf.ances is more doubtful. This report
summarizes a study of the capabilities of
the methods based on phenol reagents of
Fciin's type. Objectives of this study were
fo improve understanding of the nature of
the reactions and fo select, simplify, and
standardize a relatively reproducible pro-
cedure for the determination of fofa!
phenolic substances in plant products, p ar-
ficuiarly grapes and wines.
MATERIALS AND METHODS
Equipment: Absorption spectra were
determined with a Bausch and Lomb
Specfronic 505 recording specfrophofo-
m.efer equipped with a phofofube respon-
sive fo wavelengths up fo 750 m/~ supple-,
men+ed by a Zeiss PMQI! specfrophofo-
meter for readings between 750 and 1000
m/~. Molar extinction coefficients were fak-
,an from the Specfronic 505 curves. Blank
samples (water plus reagents)prepared un-
der ihe conditions being tested were used
as reference (zero absorbanoe) samples,
but in all cases tested there was very liffle
or no difference in absorbance over fh:e
visible negion between these blanks and
distilled wafer.
A transistor-regulated Bausch and Lomb
Specfronic 20 equipped with a red filter
and infrared-sensitive IP40 photofube
was used for most of fh,e analyses and
found fo be quite satisfactory, although
results were comparable with the specfro-
pnoromefers named above and with an-
I ,
other colorimefer. Some of the experiments
were conducted with 3/~-inch-diamefer fesf
;45~PHENOLICS DETERMINATION
tubes used as absorption cells, but in most
cases a I-era-light-path 0.2-ml microcell
in a Bausch and Lomb flow-1.hrough
cuveffe assembly was used in order to
minimize possible positioning errors.
Reactions af specific temperatures were
obtained w:i1.h a bath con1.rolled a1. 30.0,
40.0, or 60.0°C wi1'hin a small fraction
o f a degree. A boiling-wa1.er bath was
used for 100°C 1.es1.s, and 23.5-Z-_0.5°C
was~the laboratory room 1.empera1.ure.
Reagents: All organic subs1.ances, unless
otherwise sfafedi were commercial mater-
ials equivaienf or be1'fer t-o Eas1.man white
label in purify. All inorganic reagen1.s were
anatyficai reagen1' grade. Gallic acid was
decolorized wi1'h ac'1'ivafed charcoal and
recrysfallized 1'hree times from aqueous
solution. This gallic acid, 1.annic acid, and
d-calechin were dried for s,everal hours af
80°C in vacuo over phosphoric anhydride
to produce the a nh(/drous ma1'erial. The
o1'her materials were assumed 1'o contain
1.he wafer of crysfalliza'fion indicated on
flne labels.
Soiufions were prepared and dispensed
a1' room iempera1.ure by s1.andard volu-
me1.ric techniques. Unless 1'he desired con-
central'ion co,lid not be reached, all solu-
1.ions were prepared in dis1.ili,ed wafer. Sub-
s1'ances, such as querce1'in, which were not
soluble in wafer were dissolved in 1'he
minimum proportion of efhanoi plus wafer
necessary 1.o give comple1'e solution a1'
about 400 mg/li1'er.
Folin-Denis reagen1' was prepared ac-
cording 1'o s1'andard directions (I, 7) ex-
cept 1.ha1. we were unable "1o obtain con-
sisfanf results from 1.he reagen1.s prepared
by digestion on a si-eam bath, and 1.here-
fore used refluxing conditions.
kolin-Ciocal1.eu reag,ent-is prepared es-
sentially as originally described (2): 100 g
of sodium 1.ungsfa1'e (Naz'vVO42H20) plus
25g of sodium molybdafe (Na2MoO42H20)
are dissolved in 700 ml of distilled wafer in
a 2-1i1'er round-bof1'omed pyrex flask, 50 ml
of 85% H3PO4 and 100 ml conc. HCI
are added, and a reflux condens.er is al-
l'ached via an ungreased ground-glass
joint. The solution is refluxed 10 hr (not
necessarily conl-inuous in our experience).
A few glass beads helped suppress bump-
ing, bui- some trouble was still experienced
and if was found best To angle the con-
denser so "l-hat the solution would be unable
fo spurt straight out vertically and fo heat
fh,e flask strongly with an open bunsen
flame directly on the wire-gauze flask sup-
port. Boiling chips other than glass disin-
tegrated and contaminated the solution.
Af the end of fh:e refluxing period, ,heat-
ing is stopped. Distilled waf,er, 50 cc, is
us,ed fo rinse down the inside of the con-,
denser and lower the temperature safely
below boiling.
The condenser is removed, and 150 g
of lithium sulfate (Li2SO4H20)added. Tak-
ing all precautions against breathing the
toxic vapor, add a f;ew drops of Br2 and
reheat the solution and boil for 15 min
without a condenser fo remove the excess
bromine. The bromine oxidizes any traces
of molybdenum-tungsten blue, and the final
reagent should be yellow without any green.
A small amounf of 30% .hydrogen peroxide
can be substituted for ihe Brz if a hood or
other safely v,enfilafed area is not avail-
able (14). The solution is cooled, made fo
one liter, Filtered through sinf,ered glass
if no I- completely c!ear, and stored, prefer-
ably in a gbss-sfoppered amber bottle.
The prepared solution may be purchased
from laboratory suppliers.
Procedure: The reaction conditions used
as ihe basis of comparison for most of
these studies are nearly the same as pre-
vious standard methods (I, 7). Samples of
appropriate size such as I ml of white
wine are added fo about 7S ml of wafer
in a 100-ml volumetric flask. The Folin-
Denis reagent, 3.0 ml, is added and mixed,
foJlowed by 10.0 ml of an aqueous solution
containing 20 g anhydrous NazCO3 per
100 ml, and the flask filled fo the mark
with wafer. The contents are mixed, and
after an hour fh,e intensify of the blue
color is measured (at 675m/.J in comparison
with standards prepared similarly with a
known phenolic compound.
RESULTS AND DISCUSSION
Folin-Denis procedure and '"i'annin" de-
termination. Prior 1.o adoption of the Folin-
Denis colorimefric procedure for "1.annin"
in wines and spirits, the official A.O.A.C.

m.efhod involved titrimefric oxidation of
dealcoholized wine with permanganate
soluf:,on before and after treatment with
carbon. The difference between the two
~esulfs was described as tannin plus color.-
incj matter. If was clearly recognized that
more than true tannin in either the macro-,
molecule, leather-tanning, or astringent
flavor sense was included in the value ob-
rained unless special fracfionating pro-
cedures were employed (8, 10, 17).
This useful distinction fended to be-
come biurred when the Folin,-Denis pro-
cedure was adopted as the official method
for "tannin" in wines and spirits. This color~
forming reaction is produced :by monohy-,
dric phenols, polyphenols~ flavonoids, tan-
nins, and some other readily oxidized sub-
stances such as ascorbic acid. Thus, if
appears preferable fo us to term the value
obtained "total phenolics" rather ihan
"tannin." As a method for total phenolic
estimation the optimum conditions would
be those which give the mosi complete
reaction and reproducible results with all
phenols present with the least interference
from other substances,
Smif ef a/. (17) compared the per-
manganate procedure with the Folin-Denis
procedure in analyzing a series of purified
tannin preparations and fruit samples. Their
results illustrated the empirical nature of
both methods. There was only approximate
agreement between the quantitative va!ues
obtained by the two methods upon the
different types of purified tannin, but the
values calculated for the fruit samples by
the two methods agreed quite well. From
these and other data (e.g. 15, 22) if is
clear that .approximately the same value
expressed as tannic acid content is obtain-
ed with the Folin-Denis method and the
Neubauer-Loewenthal (permanganate)
m,ethod when they are properly applied
to fruit, grape, wine, and spirit samples,
Reactions involved. The ,active reagent
consists of a yellow acidic solution contain-
ing complex polymeric ions formed from
phosphomolybdic and phosphofungsfic
heferopoly acids. Apparently this solution
contains an infergraded polymeric series
having the general form of a central tetra-
hedraJ phosphate unit surrounded by
PHENOLICS DETERMINATION--146
several ocfahedral molybdenum oxy-~.cid
units in which structure tungsten can freely
substitute for molybdenum (9). This reag,enf
oxidizes phenolafes, and the heferopoly
acid becomes partially reduced from the
-~.6 to a mixture of -!-6 and -t-5 valence
states, resulting in the production of com-
plex molybdenum-tungsten blue. Complete
reduction fo the lower valence state des-
troys the color. The exac'f nature of these
complex blue pigments is still uncertain,
but many of their properties and their
suitability for colorimetry .have long been
known (9, 24).
The phenols are oxidized rapidly only
in solutions sufficiently alkaline to give ap-
preciable concentrations of the phenolate
ions. Unfortunately the oxidizing reagent
and the blue pigment formed are unstable
in alkaline so!ufion. Competing reactions
involve destruction of the active yellow
re.agent by alkali, ionization of the phenol
by aikali, reduction of the reagent by the
phenolate to produce the blue product,
and destruction of the blue pigment by
alkali, if is not practical, owing fo rapid
destructive reactions, fo raise the pH to
the point that all the phenols are com-
pletely converted to phenolate ions. Most
phenols are about 50% ionized af pH
9-10. As the ionized portion reacts with
the Folin re,agent the equilibrium will shift
and more ph,enolafe be produced. Time
wouid be required for this reaction fo ap-
proach comp!etion. 'In order that some
Folin reagent survive the alkaline condition
long enough to react with all the phenolafe,
a considerable original excess is desirable,
particularly for samples with high phenol
content.
From these considerations it would ap-
pear that the optimum conditions for
reasonably rapid I~roduction and a rela-
lively long retention of maximum color
would include a high level of the phospho-
molybdo-tungsfate reagent and a moder-
ate alkalinity.
Folin-Denisphenol reagent. The original
paper by Folin and Denis (3) described two
color reagents" a uric acid reagent (with
which we are not concerned here), and a
more general pheno! reagent. Af about the
same time they reported the use of their
147--PHENOLICS DETERMINATION
Folin-Denis phenol reagent fo quanfifafe
fyrosine in proteins and vanillin in van'lla
extracts (4, 5). Scoff (I 6) applied this
reagent fo the determination of phenolic
substances in alcoholic beverages, and the
re..~drion was studied and improved by
Vala,er (22, 23) Rosenblaff and Peluso (15),
and Pro (!1, 12, 13), culminating in the
official or Folin-Denis-Pro procedure (7)
also included in the Uniform Methods of
Anaiyses for Wines and Spirits published
by the American Society of Enologisfs in
1963.
The significant parts of this procedure
were fh,e use of I ml of sample, 5 m! Folin-
Denis reagent, 10 ml saturated sodium
carbonate solution as the alkali, 100 mi
final volume, a 30-minute color develop-
menf period, measurement af 650 fo 76.0
m~, and a tannic acid comparison stand-
ard. A good degree of reproducibility
and accuracy have been reporfect for this
method from individual and collaborative
sfuclies (11, 12, 13, 15, 22, 23). However,
several feafures can cause inconvenience or
possible error. Saturated sodium carbon-
ate varies in composition with temperature,
and equilibrium is not reached rapidly
from the super-saturated condition. Beer's
law may not Be followed over a sufficiently
wide range, and in particular we have oh-
rained abnormally low results if samp!es
of too high a'bsorbance are diluted with
a blank solution insfeacl of repeating the
entire assay procedure after further dilu-
lion. If samples are inad,equafely m'xed
Between additions or if reactants are
added in the wrong sequence, results are
erratic. Depending upon femperafur, e and
other factors, the blue color may be form-
ing or disappearing af an appreciable
rare after 30 minutes and produce ,erratic
results or requine a rigid time schedue
difficult fo m~infain with a large group of
samples. And, most serious of all a pre-
cipitate: may form slowly and will affecf
results unless noticed ancJ removed.
This precipitate is a white, dense,
crystalline material which is formed in
greater amount in the presence Of incree.s-
ecJ concentrations of either the Folin'Denis
reagent, the sodium carbonate, or both
(as with less original dilution wafer than
the specified 75 ml) as shown in fab e I.
Less precipitate is produced as a rule
(S,efsC and D Table I)in samples produc-
ing more blue pigment (containing more
phenolics). The Drecipifafe may occur er-
rafically, depending upon the manner of
mixing (Set B, Table I). If the precipitate
is removed by cenfrifugafion the blue
color yield is in most cases the same as
expected from samples prepared so fhaf
the precipitate did nofform,'buf filtration
through paper leads fo adsorption of the
blue pigment and lower values, parficularlv
w'~h more dilute samples, even if the first
few ml are discarded (Table I).
-[kiis precioifafe is evidently .a sodium
salt and is cierivect from the active reagent;
therefore, its appearance in this system
can be prevented only by not exceedinq
its solubility. This means keeping the con-
cenfrafions of both the Folin-,Denis reagent
and the sodium carbonate as low as pos-
siBie. Such considerations have ied fo use
of !essoffhesereagenfsfhan in fhe official
method (i), but maximum color then de-
ve!ops more slowly and Beer's law will
not be {oJlowed fo as high a phenolic
content.
Folin-Ciocalfeu reagent. The original
reports by Foiin and Denis (3, 4, 5) were
followed by a long series of papers by
Foiin and his co-workers and by others
noiing problems and suggesting improve-
menfs in these methods. Modifications in
the phenol reagent itself cuiminafed in
the report by Folin and Ciocalfeu (2). The
modifications consisted mainly in a onger
heating period, the presence of hydro-
chloric acid, and the addition of lithium
sulfate. The Folin-Denis reagent contains
two classes of substance, one more sensi-
five than the other to reduction, whereas
the F:olin-Ciocalfeu reagent has a greater
incorporation of rnolyBdafe into the corn-
plex, giving the form highly sensiiive to
reduction. (24).
The lithium sulfate is added to prevenf
precipitation of the sodium complex ~alts
(the white precipitate mentioned above).
The addition of the sulfate ion appears to
prevent this precipitation by hydrogen-
Bonding in some manner to hold the corn-
plex sodium salt in solution. We suggest
 TABLE I
Precipitate Formation and Removal in a Folin-Denis Total Phenolic Assay

Sefa

Gallic acid A B C D
Filtered Filtered Filtered
#g Kb K Kb__" K TurbidifyC K Kb : K TurbidityC K Kb__" K TurbidifyC

0 .054 .068 .353

94 .157 .150 1.01 .293 .157 0.86 .032 .161 0.84 .277
281 .157 .157 0.93 .003 .171 0.90 .010 .176 0.92 .204
374 .159 .164 0.96 .000 .170 0.93 .001 .182 0.94 .152
468 .164 .185 0.88 .020 .173 0.94 .010 .173 1.01 .170

a Set A - 3.0 ml Folin-Denis reagent; 75 ml dilution wafer, 2.0g Na2CO3, 100 ml final volume, after I hour af 23.5°C no precipitate formed.
Set B _- 6.0 ml Fo[in-Denis reagent, otherwise like A, precipitate formed.
Set C _- 20 ml dilution wafer, otherwise like A, precipitate formed.
Set D - 20 ml dilution wafer, otherwise like B, precipitate formed.
b K ~_ absorbance at 675 m# in 1.65 cm ID test-tube per 100 #g gallic acid, obtained after cenfrifugafion for those with precipitation.
c Turbidify -- absorbance before cenfrifugafion minus absorbance after cenfrifugafion.
 149--PHENOLICS DETERMINATION
this because we have encountered no pre-
cipifafe with the complete Folin-Ciocalfeu
reagent af any concentration of sodium
carbonate we have used except when the
solution was heated fo 100°C. Af 60°C
and below no precipitation wasencounfered
and the effect of high temperature is
attributed fo thermal rupturing of hydro-
gen bonding, if the lithium sulfate is
omitted, blue pigment is produced in nor-
mal proportion af the levels of phenol
tested, but precipitation also occurs.
Lithium is used because, of course, so-
dium adds fo fh,e sodium level and the
potassium and ammonium salts of these
complex acids are even less soluble than
the sodium salts. Sodium carbonate is
generally preferred over other alkalis which
have been used such as sodium cyanide
and sodium hydroxide (2, 2 I). This prefer-,
ence is because precipitate formation
seems fo be greaferwifh NaOH, and blue
color appears fo fade more rapidly with
NaCN than occurs with Na2CO3. Aga'n,
an effect similar fo hydrogen bonding by
the carbonate ion is suggested,
Comparison of Folin-Denis and Folin-
Ciocalfeu reagents: The l:olin-Ciocalfeu
reagent has been generally adopted as
an improvement by clinical laboratories
(for i-yrosine, trypfophan, and protein de-
terminations), and if would appear to be
advantageous for all fatal phenolic defer-
minafions. If has been stated fo be inf,er-
changeable with the Folin-Denis formula-
lion, but testing with samples of interest
fo us appeared desirable,
Pro (I I) reported the absorption maxi-
mum of fh.e Folin-Denis procedure fo be
760 m/z, whereas Swain and Hillis (21)
reported 72S m/z and analytical measure-
ments have been made over a wide range
of wavelengths, as previously noted. We
obtained absorption spectra for the reac-
lion products between several phenols and
both [:olin-Denis and Foiin-Ciocal~eu
reagents" figure I shows typical examples,
Eolin-Denis-blue exhibited a very broad
maximum which shifted with fim,e from
7S8 m/z to as low as 700 m/~ and became
even broader and more nearly flat. The
average maxima with several different
samples were 734 m/~ af I hour, 726 m/z
af 5 hours, and 721 m/z af 6 hours.
F:olin-Ciocalfeu-blu,e absorption was con-
siderably more intense than in samples
prepared similarly with Folin-Denis reaaenl-,
except af the shorter wavelengths (Figure
I). Wiih gallic acid samples fh,e absorb-.
ance ratio F-C/F-D was 1.10 af 660 m/z,
1.10 af 700 mff, 1.21 af 750 m/z, and
about 1.35 af 765 m/z. The maximum with
FoJin-Ciocalfeu reagent was broad, but
less so than with the Folin-Denis reag,enf.
The shift fo shorter wavelengths with time
also appeared fo be smaller, the maximum
becoming cons'rant af about 765 mff with-
in one hour.
Since the pigment measured arises
from reduction of the reagent, the nature
of the reducfanf (phenol) would be ex-
pecf.ed nat fo influence the absorption
spectrum qualitatively. However, the pres-
ence of a reactive and a less reactive form
of the oxidant in the Folin-Denis reagent
rr,ighf produoe, a d'fferenf mixture of blue
substances with different subsfrafes. The
relatively sharp maximum anal more sym-
mefricai absorption spectrum with Folin-
Ciocalfeu reagent reinforces this sugges-
lion. The relative standard deviations of
the ratios of 'the absorbance at different
wave!engfhs were determined for each of
eight different ph.enols treated with either
i:olin-.Denis or I:olin-Ciocalfeu reagents
and spectra determined after I, 4, S, and
6 hours af room temperature. The results
indicated fhaf the sp,ecfra obtained with
different phenols were quite similar with-
in the group reacted with either of the two
reagents, but there was sightly greaf, er
variability in relative absorbance af dif-
ferenf wavelengths with the I:olin-Denis
set than with the F:olin.-Ciocalfeu set. In
most cases variability was I,ess af four
hours than af ~he other times tested.
Table 2 shows the molar absorptivity oF
a series of phenols and other easily
oxidized substances after reaction with the
two reacjenfs under the same conditions (S
mi reagent/100 m! solution, I hour at
23.$eC, 72S m/z). No substance tested
produced reaction with one reagent and
noi i-he other. The Folin-Ciocalfeu I~eagenf
produced more blue pigment than did
the Folin-Denis reagent with all phenols.
 TABLE 2
. .

Color•Yield with Folin-Ciocalteu and Folin-Denis Reagents with Various Phenols and Other
Reducfanfs on a Molar Basis and Relative to Gallic Acid

Molar absorptivity
af 725 m # - - l O 0 0 Gallic acid equivalenfb
Folin-Ciocalfeu value-y-Folin-
Liferaturea Denis value, x 100
Folin- Folin-
Ciocalfeu Denis FC/FD Folln-Denis pKa 660 m# 700 m# 725 m# 750 m#

Phenol 11.6 9.1 1.28 9.9 100 II0 106 106

Phloroglucinol 11.2 8.7 1.29 7.7 106 114 107 107
I-Na.phfhol 12.2 10.3 1.18 3.7 96 102 98 98
L-Tyrosine 15.5 12.5 1.24 I0.1 99 105 03 102
Vanillin 14.0 9.2 1.52 7.4 123 132 26 125
Salicylic acid 4.8 2.2 2.17 13.4 207 212 79 205
4-Mefhylumbelliferone 7.8 3.4 2.34 203 210 94 183
Coumarin 0.8 0.4 1.77 235 191 46 167
Cafechol 21.5 17.1 1.25 20.2 9.4 99 109 04 105
Pyrogallol 20.9 16.1 1.30 10.9 102 113 07 108
Gallic acict 23.3 19.3 1.21 18.5 100 100 O0 I00
Tannic acid 186.7 152.7 1.22 10.9 95 105 OI 102
Grape seed extract 92 99 96 96
d:Cafechin 34.5 26.8 1.28 30.7 104 II0 106 107
Quercefin 56.9 41.7 1.36 105 119 113 112
Ascorbic acid 15.1 14.0 1.08 89 94 89 .91
Fe(NH4)2 (S04)2 5.4 4.9 I. I0 104 105 91 90
Na,S~O~ 0.6 0.6 1.00 91 100 83 80

a pK~ from sfanclard handbooks, molar extinction coefficients from Swain and Hillis (21).
b Gallic acict equivalent was caiculafed by multiplication of the absorbance oi: each sample by a single value of gallic acicl weight per absorbance unit
determined under the same conditions.
1 5 1 - - P H E N O L I C S DETERMINATION
Note, however (Table 2), fhaf the pro-,
porfionafe color yi,eld was relatively poor
for some of the phenols such as vanillin
or 4-mefhylumbelliferone with the Folin-
Denis ~agent. Not only did the Folin-
Ciocalfeu reagent give greater color than
fh,e Folin-Denis reagent with all phenols
and particularly some of the less respon-
sive ones, but if also gave slightly less
color in proportion fo fh.af from Folin-
Denis for f.he possibly interfering reducf-
ants ascorbic acid, ferrous ion, ancl sulfur
dioxide. Thes,e effec:fs are affribufecl to the
relatively high content of active oxidant in
the Folin-Ciocalfeu preparation which pro-
motes the more complete oxiclafion and
measurem,enf of the more slowly reacting
phenols. The interfering substances are
less limifeol fo alkaline re.action conditions
and are more easily oxidized than some
phenols, which probably explains their dis-
proportionate reaction with the weaker
Folin-Denis reagent,
.6 i 1
.S
iii
U
Z
.4
0
nm
.2
.I i i
500
WAVELENGTH, mp
~igure I. Absorption spectra produced by wine (W) and gallic acid (G) with Folin-
Denis (D)and Folin-Cioc~lfeu (C) reagents.
Table 2 shows the comparison obtained
by faking the gallic acid sample as a refer-
ence and calculating the mg of gallic acid
equivaienf in absorbance at various wave-
lengths for the different other materials
after re.~cfion with each Foiin reagent. The
ga;lic acid equivalent with the Folin-
Ciocalfeu reagent was about 96-114~"o of
the Folin-Denis vaiue for the readily re,-
acting phenolics at ali four wavelengths
examined. The less reactive phenolics such
.as salicylic acid showed the expected
greater response with the Folin-Ciocalfeu
reagent, and the nonphenolics showed
slightly less response. These effects were
apparently not as uniform By wavelength,
and, again, this is affribufect to the effect
of the two types of reactive substances
in the Folin-,Denis reagent.
From all these data the conclusion is
drawn fhaf fh,e Folin-Ciocalfeu reagenF
is preferable fo the Folin-Denis reagent
for several reasons. A.Ifhough the pigment
1 i i I
WC
GC
L
GD
I I I
600 700 800

obtained under the same conditions is
consict,erably greater (and therefore ana-
lyficaily preferable)with the Folin-Ciocalfeu
reagent, comparison with a standard
phenol should produce good agreement
with comparable values obtained with the
Folin-D,enis reagent unless a high propor-
lion of less reactive phenols or interfering
reducing substances other than phenols
was pros,ant. In ]his event the Folin-
Ciocalfeu reagent would be expected fo
give the better estimate of total phenolic
content.
Choice of a reference-sfandard phenol:
The molar absorpfivifies given in fab!e 2
re-emphasize fhe empiricaJ nafure of fhe
mefhod as applied fo a complex mixfure,
However, fhe dafa appear fo b,e more
easiiy rafionalized fhan nofed previously
(23). The monohydric phenols, from
our own dafa and dafa of Swain ef a/.
(6, 20, 211, produce molar absorpfivify of
-I-he order of 11-14,000. Those fhaf pro-
duce appreciably less fhan fhis appear fo
be less reacfive owing fo subsfifufion,
which raises their phenolic pKa appreciably
(salicylic acid)or imposes a masking or-
sferic effect. Coumarin might be consider-
ed as an ,extreme case of the !after--if
has no free phenolic group, but presum-
ably the lacfone is partly hydrolyzed b,/
the alkaline medium, giving some phenol
reaction. The surpression of reaction of
the 7-OH group in the umbelliferone
derivative could conceivably result from
the decreased bond conjugation possible
in an oxidized quinoid form than in the
r,educed form. The same reasoning seem~
appropriate in the mefa-polyphenols. One
hydroxyl could oxidize fo give a reasonaf-
ing quinoid structure, bui more would not.
Phiorog!ucinol reacts as a monohydric
phenol in our tests, and the resorcinos
h,~ve also appeared roughly similar fo
monophenols (20, 2 I).
Cafechoi reacts as if both hydroxys
were oxidized, presumabiy fo give the
orfhoquinone, and gives double the molar
color yield of phenol. Both gallic acid and
pyrogal!ol reacted in our 'rests as if two
but not fhre.e hydroxyls were involved, as
would be predic+ed. The molar absorptivity
of d-cafechin closely approximates fhaf of
PHENOLICS DETERMINATION--152
phioroglucinol plus cafechol, as would be
predicted. Of course, a direct sfoichio-
me'frv cannot always be visualized because
of f{le interdependence of several com-
p,efing reactions and the specific reaction
conditions.
Under these circumstances any normally
reactive and otherwise convenient phenol
would appear fo be suitable as a com--
parison standard for colorimefry of total
phenolics with the Folin- Ciocalfeu reag,enf.
Tannic acid has been commonly used (7).
Tannic acid is glucose-penfagalloylgallafe,
i.e., if confains fen pofenfial gallic acid
moieties. From fhe above considera'fions if
would be predicfed fhaf (fwo oriho OH
being free in each moiefy) pure tannic
acid would give fen limes fhe molar color
yied of gallic acid. Table 2 shows lhaf
pure gallic acid gave a molar absorpfivify
of 23,000 compared fo 187,000 for our
fannic acid sample. This agreem,enf wifh
fhe expecfed value is considered fo be
good considering possible impurifies in fhe
fannic acid.
23.5°C
.32
7 ~ ~.~ c
.30
f - ~ ",o,,.......~_ 40 °¢
u.I *"o
u .2s-
~ .2~
.24
40°C
.22
I l
2
I
4
I ~ I
6
Z I
8.
HOURS
Figure 2. Absorbance development be-
fween gallic acid and both phenol reagents
with time af 23.5°C and 40°C with 2.0
g/100 ml (solid line) or 3.0 g/100 ml (dash-
ed line) sodium carbonate.
153---'P'HENOLICS DETERMINATION
Fortuitously, galic acid has a molecular"
weight of 170.1 g and pure tannic acid
is just 10-,fold as large, 1701 g.
Th,erefore the same numerical value should
be oBfainecl whether the results of a total
phenol assay are expressed as weight units
o# gallic acid or tannic acid. This is desir-
able in order fo produce values compar-
able with previous "wine tannin" values,
Gallic acid is a more satisfactory standard
than tannic acid because purify is easier
fo obtain, fo retain, and fo ctemonsfrafe,
Gallic acid offers satisfactory solubility,
aclequafe stability, low price, etc. Data
obfainect in the ctevelopm,enf of fable 2
and in other experiments have disclosed
no anomalies or problems in the use of
gallic acid, and if therefore appears fo
be fh,e preferable reference standard for"
fofa! phenolic assay by the Folin-Ciocalfeu
method,
Reaction procedures. Rosenblaff and
Peluso (15) found fhaf bright dayl'ghf
had no effect upon the assay, and we have
found no difference befw,een samples left
in a well-lighted laboratory versus aliquofs
kept in the dark. They found no difference
between samples exposed fo fh,e air in
different fashions. Air contact has appear-
ed fo make no difference in our sfudie_~. In
fact, color development and rei~enfion in
a!iquofs through which 02 or N2 gas
(saturated with wafer vapor) was passed
af 100°C gave the same absorbance at
725 m~, during ihe 11/2-minute test, which
was weli beyond the period of maximum
color dev,elopmenf af this temperature,
Pro (11), as well as Rosenblaff and Peluso
(15), investigated the amount of Folin-
Denis reagent fo give maximum color, and
found fhaf 5.0 ml per 100 ml of fina!
colored solution was more than sufficient
for maximum color development over th.e
necessary range of phenol content. The
Foiin-Ciocalfeu reagent contains a con-
sicterably greater content of active oxidant
than the Foiin-Denis reagent, but, since
a great excess is desirab!e and precipi-
ration is no longer a problem, the same
proportion was refaineci without furf~her
fasting. Those workers have also shown
that the conoenfrafions of phenol, oxi-
clanf, and alkali interact with temperature
fo produce variable times during which
fhe color being measured is maximal. Maxi-
mum color is reached in less time, of
course, with more Folin reagent, less
phenol, more alkali, and higher tempera-,
fure. The blue color, once produced, is
more stable with lower alkali and lower
femperafur, e. We performed several addi-
fional experiments of the type illustrated
in figure 2 in order fo estimate the rnosf
convenient and reliable combination of
conditions. Iviosf of ihese experiments were
performed with ga!lic acid samples at 0.2
fo 0.3 mg/100ml of final colored solution,
but ~esfs with wine samples and with gallic
acid a • other conoenfrafions ind"icafea'
fhaf the results could be generalized.
Approximately the same maximum absorb-
ance value (within the reproducibility of
replicates) was obtained with samples con-
raining the same amount of phenol over
most of the range fasted (room fempera-
lure fo 100cC, 2.0 fo 3.0 g Na2CO3 per
100 ml).
The Arrhenius relationship appears valid
for these data in fhaf the log of the time
fo reach maximum color af a oerfain re-
action composition was proporfiona! fo
the reciprocal of the absolut-e fempera-
lure. Figure 3 summarizes the data over
the range which appears fo be most useful.
Data obtained outside fh,e limits shown
were included in the estimate of the best
straight-line approximation for the time
and temperature for maximum color de-
velopmenf and retention of this color
within the typical variability of replicates
(about 0.003 absorb ance).
Figure 3 indicates fhaf the color deve-
opmenf times previously recommended are
net long enough if room i~emperafure is
relatively cool and one is fo avoid read-
ing the samples during a period of ap--
preciable color change. No one set of
conditions appears ideal for all possible
ambient temperatures, 'but a suitable com-
binafion can be selected from figure 3.
Table 3 gives the approximate tempera-
lure coefficients of the color-forming and
fa.-Jing reactions. From these data if ap-
pears that temperature has a siighfly
smaller effect with the higher sodium car-
bonafe content, without evident disad-
 PHENOLICS DETERMINATION 154-

vantage in terms oT Tading. If many samples are being prepared or if

For our purposes, a color develop- ambient temperature is higher, it would
menf period oT 2 hours af room fem- be advantageous to lower the carbonate
perafure (75°F) in the presence oT cotent and adiusf the time {or reading
3.0 g Ikla2CO3/100 ml appears desirable, accordingly, so that small variations in

10
/S /
# / .# /~"

$°ssS .iS $~~ 2A 2.5A

-~ 3
/'
111
u
z~

t~
,,,, /./,', F
o "r 2
e $
t
I
S
//,'//o

t/s I
Q5
50 40 30 20 ]O°C
(]22) (]04) (86) (68) (50)oi:
TEMPERATURE
Figure 3. The time and temperature relationships Tor the initialdevelopment of maxi-
m u m color (A) and onset oT appreciable Tading (B)with gallic acid reacted with
Folin-Ciocalfeu reagent in the presence oT 2.0, 2.5, and 3.0 g/100 ml oT sodium
carbonate.
~55~PHENOLI'CS DETERMINATION

timing will not become a source of de-
creased reproducibility. If a controlled
bath is available, a precise s,electecl per-.
iod af a regulated warmer temperature,
foiiowed by holding of the samples at a
lower temperature unfiJ they can be read,
should give the best combination of rapid
color formation without necessity for pre-
cise timing of the preparation or reading
of the samples.
Th,e Beer-'Lamberf law is not strictly fol-
lowed over a wide range of phenol con-
font, because the amount of phenol pres-
ent influences the time necessary to reach
maximum color, etc. The deviations are
very small, however, and the standard
curve is quite reproducible. In one series
of tests with 5.0 ml Folin-Ciocalteu
reagent, 2.5 g Na:CO3/100ml, and room
temperature, the K-.valu,e (absorbance per
100 /~g gallic acid/ml colored solution, af
765 mt~, I-cm light path) was 0.138 af
.82 ffg/ml and 0.151 af 4.10 ffg/ml, and
0. i36 af 12.30 ffg/ml after 4!/2 hours. The
latter sample was clilufed I/3 with blank
in order to be read on the colorimefer.
This procedure extends the standard curve
and is very convenient when unknown
samples are unexpectedly high, whereas
by the previous method (I, 7)if was neces--
s.~ry fo run the assay again. The mean
K-value and relative standard deviation
as ~/'o of the mean for the nine samples
oF gallic acid covering the whole range of
0-1230 fig/100ml, was 0.141+4.45°/_,,o after
2 hours, 0.145±4.14% af'fer 3 hours, and
0.143 -.L:3 .99 % after 4!/2 hours.
The timing of the addition of the
reaaenfs can be important. Swa;n and
Hillis (21)em.phasized fhaf the carbonate
should be added exactly 3 rninufes .after
acldifion, of the Folin peagenf. Our experi-
ments indicated that, with sample plus
75 ml dilution wafer plus 5.0 ml Folin-
Ciocalfeu solution and then addition of
the carbonate, 25-30 seconds between the
Folin peagenf and the carbonate gave
slightly higher (2.5-6%)and .more erratic
absorbance but no difference was found
over the period I minute fo 8 minutes.
However, it is important fo mix the sample
and the Folin-,Ciocalfeu reagent under
dilute conditions before addition of the
carbonate, which may be relatively con-
centrafed. If the sample and F-olin reagent
are mixed and then diluted, the absorb-
ance is again higher and less reproduc-
ible. If 60°,/o or more of the final volume
is present with the sample befope addition
of the Foiin reagent or if the Folin-
Ciocalfeu reagent is diluted fo this pro.-
portion Before ,addition to the sample,
reproducible results have been obtained.
IXlo difference in the absorbance produced
was defected with samples of Folin-
Ciocalfeu reagent which had been diluted
to I / i 2 its original concentration for p,er-
ieds of af least 12 hours at room tempera-.
lure before use.
Substances likely fo 'be present in wines
and grape samples which might affect
the apparent phenol content include sugar,

TABLE 3
Approximate Temperature Coefficient of the Color Reaction Between Phenols and
Folin-Ciocalfeu Reagent in the Presence of Sodium Carbonate.

Time ÷o reach max. color at Time to fade delectably

higher temp -~- lower femp at higher femp -~- lower femp
Temperatures
2.0a 2.5 3.0 2.0 2.5 3.0

20°__30°C 2.7 2.5 2.3 2.7

30°__40°C 2.6 2.5 2.2 2.6 2.6 2.6
40 °--50 oC 2.5 2.3 2.0 2.4 2.5 2.4

a Grams Na2CO3/100 ml.


alcohol, and farfaric acid. Addifion of up
i-o 2 ml of efhano! per 100 ml of final
colored soiufion did nof noficeably affecf
fhe absorloance produced wifh gallic acid.
Tarfaric acid (I ml of 1.5% solufion added
fo a 100 ml gallic assay sample) did nor
affecf fhe absorloance obfained eifher,
bur I mi of a 20% glucose solufion in-
cpeased ihe apparenf phenol confenf
abouf 10°//o from 340 /~g fo 382,~g • O~,her
oxidizable subsfances which woud inferfere
(see Table I) include ascorbic acid, hi-
sulfife, and ferrous ion. Af fhe leves ordin-
arily presenf in wines and grape samp!es,
fhese would nor confribufe a greaf deal
of appar.enf phenol, and fhey can be
defermined separafely and a correcfion
made if fhaf appears imporfanf for par-
ficular samples,
The imp,roved mefhod-precision and ac-
curacy. Based upon fhe sfudies described,
fhe procedure selecfed consisfs of mixing
I,-ml samples (usually as is for while wines,
1/10 dilution for red wines) wifh af leasf
60 ml of wafer in a 100-ml volumei-ric
flask. Folin-Ciocalfeu reagenf, 5.0 ml, is
added and mixed, and affer abouf 30
seconds and before abouf 8 minufes 3.0 g
of anhydrous NazCO3 in aqu,eous soln. (e.g.
15 ml of 20°./'o soln.)is added and mixed,
and fhe confenfs of fhe flask made fo
-I-1
volume, i ne absorbance is defermin,ed
versus a zero-,absorbance reagenf blank
afier 2 hours af 75°F, preferably in I cm
cells af 765 m/~. The fofal phenolic con-
fenf is calculafe/ in gallic acid equiva-
I,enfs by comparioon wifh a sfandarcI curve
similarly prepared wifh zero f o abouf
500/~g gallic acid per 100-ml flask,
This improved procedure was compared
wifh fhe procedure formerly in use in fhis
labor afory (Procedur~e--Maferials and
Mefhods) wifh regard fo reproducibilify
wifh a series of wines and +he recovery of
phenol added fo wine. Tesfs were also
made fo show fhaf fhe improved pro-
i
cedure could Be reduced in scale fo a
final volume of 20 ml wifhouf prohibifive
decrease in reproducibilify. In fhis modi-
ficafion we used 2.00 ml of a more dilufe
sample (I/10 fhe concenfra-l-ion for i-he
100-ml version), 10.00 ml of a 1/10 aqueous
dilufion of Folin-Ciocali-eu reagenf, and
PHENOLICS DETERMINATION--156
8.00 ml of wafer confaining 0.6 g o1:
anhydrous NazCO:~ (75 g/I). The ofher
condifions remain lhe same excepf fhaf
fhe final volume is 20.00 ml wifhouf adjusf-
mGni. This procedupe saves on expensive
glassware andreagenfs, and, alfhoughmore
skill and care is required, lime per analysis
can also be decreased, especially if ad-
ve,nfage is faken of fhe newer fypes of
semiaufomafic dilufers and dispensers.
Table 4 shows resulfs obfained from
fwelve separafe assays wifh fhe fwo Basic
procedures, and four wifh "l-he l/5-scale
improved procedure. If can be seen fhaf
fhe sfandard curves wifh gallic more nearly
followed Beer's law (as shown by ihe
more nearly uniform K values) wifh lhe
improved mefhod. AIso,'fhe Folin-Ciocalfeu
improved procedure was aboul- one,half
as variable as fhe former Folin-Denis
mefhod, as shown by fhe smaller sfandarct
devial-ions of fhe K values and ihe smaller
relal-ive siandard deviations of fhe wine
values. The average fofai phenolic values
as mg of gallic acid per lifer of wine
agreed very closely in all cases excepf
fhaf fhe swe.ef wines were higher By i-he
o der mefhod. This illusfrafes fhe value of
fhe relafively low response of fhe Folin-
Ciocalfeu improved procedure fo infer-
feting subsfanoes such as sugar.
Thereduced.-volume[mproved procedure
was very safisfacfory and gave values
idenfica l,, for pracfical purposes, wi'fh t-he
larger-volume procedure. The variabiiify
appeared even lower for fhe reduced-
volume procedure, bur fhis is affribufed
fo fhe facf fhaf fhe four complefe assays
by fhis mefhod were simple replicafes pre-
pared af fhe same fim,e. The larger-,volume
assayswere prepared in pairs of one I::olin-
Denis "old '~ and one F:olin-Ciocalfeu "new"
sef. The fwelve assays were prepared by
iwo analysfs over a period of one week,
using af leasf fwo differenf Iofs of each
reacfanf excepf fhaf ih,e same purified
gallic acict was used. ....
The recovery of added gallic acict is
quire accurafe wifh bofh mefhods if suf'
ficienf values are averaged. The recoviei~ies
were defermined by adding known high
and low levels of gallic acid fo each wine
and defermining f h,e added amouni-By
157--PHENOLICS DETEBMINATION

TABLE 4
Reproducfibilify and Recovery of Added Phenol in the Assay of Total Phenolics
with the Former and Improved Procedures

Method
Gallic acid standard
Former Improved Improved I I/5)
#g/sample K(Abs/100 #g) K(Abs/100 #g) K(Abs/100#g)

50 0.760 4- .14 .324 -- .06 1.25 ----- .01

125 0.797 4- .08 .362 --+ .00 1.44 4- .03
255 0.858 4- .06 .428 4- .06 1.48 4- .02
380 0.943 4- .04 .428 ----- .04 1.47 -- .01
505 0.954 4- .06 .415 -- .04 1.47 ± .01
all samples 0.862 4- .187 .391 4- .056 1.42 -4- .09

mg gallic acid mg gallic acid mg gallic acid

Wines eq uivalent eq uivalent eq uivalenf
per I000 ml per I000 ml per I000 ml
dry white, Thompson Seedless 245 ( -- 7%) 232 (4- 3%) 230 (4- I%)
sweet white, Thompson Seedless 362 (4-- 8%) 307 ( ± 3%) 308 ( + 2%)
tawny port, Tinfa Madeira 93s (_ 12%) 770 (-- 3%) 741 (4- 2%)
dry red, Zinfandel 3188 (.+_ 6%) 3197 ( ± 4% ) 3177 (----- I%)
dry red, Pinof noir 2130 (4- 6%) 2170 (--4-_ 3%) 2082 (----- I~/o)
dry red, Calzin 2365 (----- 8%) 2247 (+--- 5%) 2210 (----- 0%)
dry red, Carignane 1421 ( + 9%) 1416 (+-_ J%) 138s (_+ i%)
Recovery of gallic acid 97.1 4- 28.5% 100.5 + 11.4%
added fo wine

subtracting the value for a separate assay ACKNOWLEDGMENT

of the wine alone. This throws all the vari-
ability of an individual standard curve and
an individual wine sample into the per-
cenfage of gallic acid recover~ed. This ac-
counts for the high standard deviation of
these recoveries, bu'f, again, the improved
procedure is much better than the former.
SUMMARY
Several details of the assay of total
phenolic substances have been i n v e s t i g a t e d
and an improved procedure developed,
The improvements include fh,e use of Folin-
Ciocalfeu reagent rather than the Folin-
Denis reagent, gallic acid as a reference
standard, and a more reproducible time.-
temperature color d e v e l o p m e n t p e r i o d .
The values obtained are less subject
fo variation and interference from several
nonphenols, yet are directly comparable
fo the "tannin" values obtained by fh~
previously standard method.
The Wine Advisory Board is i-banked
for funds partially supporting this work.
LITERATURE CITED
I. Amerine, M. A. Laboratory procedures for
enologisfs. Department of Viticulture and
Enology, University of California, Davis. 130
pp. (1960).
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3. Folin, O. and W. Denis. On phosphofungsfic-
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. . . .
5. Folin, O. and W. Denis. A new colorimefric
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6. Goldsfein J. L. and T. Swain. Changes in
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PHENOLICS DETERM INATION--IS3
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