American Journal of Anal yt ical Chemistry, 2011, 2, 840-848
doi:10.4236/ajac.2011.27095 Published Online November 2011 (
Copyright © 2011 SciRes. AJAC
The Influence of Initial Carbonate Concen tra tio n o n th e
Folin-Ciocalteu Micro-Method for the Determination of
Phenolics with Low Concentration in the Presence of
Methanol: A Comparative Study of
Real-Time Monitored Reactions
Nunzia Cicco1, Vincenzo Lattanzio2
1Institute of Methodologies for Environmental Analysis, National Research Council, Tito Scalo, Italy
2Department of Agro-Environmental Sciences, Chemistry and Crop Protection, University of Foggia, Foggia, Italy
Received August 17, 2011; revised September 19, 2011; accepted October 8, 2011
During the Folin Ciocalteu (F-C) micro-assay for the determination of phenolics in the presence of methanol,
fine solids can form. In a previous paper, we hypothesized that the interference from alcohol on the F-C re-
action can be minimized depending on the particular procedure used to reach the alkalinity condition. In or-
der to demonstrate our hypothesis we studied, by spectrophotometrically monitoring, the time-behaviour of
the reactions carried out in the presence of different methanol concentrations at the same alkalinity condition
from two protocols. The results showed that the interfering effect of methanol on the F-C micro-method can
be affect and even prevented depending on working conditions. In particular, the formation of fine solids can
be delayed, slowed down and prevented depending on the initial carbonate concentration used. We have ex-
plained why the initial carbonate concentration, used to reach the final alkalinity condition, plays an impor-
tant role in the F-C reaction carried out in the presence of methanol. Moreover, the results from real-time
monitoring showed that, differently from traditional F-C procedure, our procedure allows us to carry out the
F-C micro method in the presence of 6% methanol, as an extreme concentration, reading the absorbance at
real time 24 min. The real-time monitoring of absorbance can be considered as a useful means to explore the
effect of other parameters on precipitate formation caused by the presence of methanol in the F-C reaction.
Keywords: F-C Micro-Method, Phenolics, Carbonate Influence, Methanol Effect
1. Introduction
Phenolic compounds, widely distributed in plants and
food plants, are thoroughly studied by different resear-
chers because of their health beneficial properties [1,2].
Many analytical procedures have been developed to
study polyphenolic compounds, in any case, a quantifica-
tion study is always undertaken as a preliminary step. Al-
though the chromatographic methodology is considered
as the most important analytical tool for the quantitative
determination of phenolics, the traditional colorimetric
methodology is frequently employed for this purpose
because of its easier availability in many laboratories.
The colorimetric method generally used is the Folin
Ciocalteu (F-C) one that consists of a rapid oxidation
reaction of phenols by using alkali, generally sodium car-
bonate, which yields an appreciable concentration of the
phenolate ions. The phenolates reduce the yellow F-C re-
active changing it into a blue pigment, spectrophoto-
metrically measured. Due to the complexity of the com-
peting reactions involved in the F-C method, reaction
equilibrium is fairly unstable and it is not easy to find the
exact conditions for the assay [3]. In order to find the
optimal conditions to accomplish the assay, only few
authors [3-6] widely studied the influence of parameters
such as temperature, reaction time, amount of F-C reac-
tive and initial as well as final concentration of alkali.
Although the optimized conditions by Singleton are gen-
erally used [3], different variations in the F-C method are
reported in literature [7-13].
However, even though this colorimetric method is of-
ten used for the determination of phenolic content in
numerous and complex samples, the many insuperable
difficulties involved bring its usefulness into question. In
fact, due to the lack of selectivity which is peculiar to
this method, some substances react with the yellow reac-
tive causing an overestimation of the phenolic content.
Moreover, an important F-C method drawback/limitation
is that under some conditions precipitates can form.
Consequently, it is necessary to resort to additional tech-
niques, such as filtration or centrifugation, which are
time-consuming and can sometimes reduce the blue col-
our yield [3].
In order to prevent the precipitate formation, Singleton
and Rossi [3] suggest accomplishing the assay at room
temperature and not exceeding the 3% concentration of
the sodium carbonate in the final mixture, although the
initial carbonate solution to be used may be relatively
concentrated (20%). Moreover, to obtain reproducible re-
sults, Singleton et al. [14] suggest not exceeding the 1%
alcoholic concentration in the final reaction mixture.
In this regard, a sample dilution is carried out to avoid
problem of precipitate formation but, sometimes, no di-
lution of alcoholic extracts can be carried out because of
their low phenolic content.
In a recent paper [7], aimed at quickly determining the
total content of phenolics in little diluted methanol ex-
tracts, we tested an F-C micro-procedure compatible
with the final concentration of alcohol equal to 4%. The
detail of our procedure is to have used 800 l of a car-
bonate solution at 5%, carrying out the F-C reaction in
the presence of 4% alkali and at 40˚C. In other words, we
used an initial carbonate solution with a very low con-
centration when compared to that used by Singleton,
carrying out the assay at a higher final carbonate concen-
tration and a higher temperature than those suggested by
Singleton. This procedure allowed us to reduce the reac-
tion time by combining together the effects of alkali and
temperature [7].
We hypothesized that the peculiarity of the procedure
used in our F-C micro-assay can minimize the interfer-
ence from alcohol.
The aim of this study is to validate such a hypothesis
and to understand why the use of the 5% carbonate solu-
tion is important for the determination of phenolics in the
presence of methanol. With this purpose in mind, we
have compared the F-C reactions carried out in the pres-
ence of different methanol concentrations and derived
from two procedures (protocols A and B). In each of
these a different carbonate concentration has been used
to reach the same alkali amount in the tested reaction
mixtures which have the same final volumes.
The F-C reactions were monitored in real-time and the
formation of the precipitate was investigated for the first
2. Material and Methods
2.1. Reagents
Experiments were performed using the F-C reagent from
Merck (1.09001), sodium carbonate (S6139) and pure
caffeic acid (C0626), chosen as a standard phenol, from
Sigma. Methanol solutions at the different concentrations
each containing 80 mg/l of caffeic acid, which is the up-
per limit concentration of the standard that can be used in
the assay, were prepared as described in the previous
paper [7].
2.2. Protocols A and B
The details of protocols A and B are briefly reported here.
100 μl of standard solution (80 mg/l or 0-blank) at differ-
ent methanol concentrations were pipetted into separate
test tubes and 100 μl of F-C reagent were added to each.
The mixtures were mixed well and allowed to equilibrate.
For protocol A, after exactly 2 min, 800 μl of the 5%
(w/v) sodium carbonate solution were added.
For protocol B, within the 2 min, 600 l of distilled
water were added and the mixtures were mixed again,
then 200 μl of the 20% (w/v) sodium carbonate solution
were added.
The absorbance time-behaviour of investigated mix-
tures, previously swirled and poured into 1-cm cuvettes,
was monitored over a period of 2 h at 40˚C recording the
absorbance data every two minutes.
2.3. Apparatus
The absorbance variations of investigated mixtures were
monitored by the spectrophotometer UV/visible ultro-
spec 4000 (Pharmacia) equipped with a 6 position water
heated cell changer.
The morphological observation of some precipitates
from both procedures was made by Scanning Electron
Microscopy (SEM Zeiss Supra 40).
3. Results and Discussion
3.1. Plan Working
In order to verify the previously mentioned hypothesis,
the following steps were taken to obtain the data used in
this study.
Copyright © 2011 SciRes. AJAC
1) The investigated reactions were carried out at 40˚C
and at 4% carbonate concentration in the final mixture,
which corresponds to that of our previous paper [7]. The
final carbonate concentration was reached using two dif-
ferent carbonate solutions. The former at 5%, already e-
mployed in our previous paper [7], was the lowest con-
centration reported in literature, the latter at 20% was the
highest concentration reported in literature [3,14].
2) Only the 20%-40%-60%-80%-100% methanol solu-
tions were investigated to obtain a comprehensive view.
Moreover, 3 replicates were carried out on each solution
for 4 times to get a fair amount of data.
3) The absorbance time-behaviour of the investigated
mixtures was monitored over a period equal to 2 h, which
we considered long enough, recording the absorbance data
every two minutes, which we considered short enough.
4) To describe the influence of the initial carbonate
concentration used (i.e. 5% carbonate of our procedure)
on the F-C micro-method carried out in the presence of
methanol, a comparative study of the mean kinetics was
made. For reasons of simplicity, the incremental ratios of
the first and second order were calculated between adja-
cent mean absorbance values. The incremental ratio ana-
lysis allowed us to determine the beginning and the end
of the phenomenon of each mean kinetic with a good
degree of approximation. In fact, even though the con-
stant time increments were not infinitely small, they were
small enough (two minutes) with respect to the two hours
of kinetics so that possible errors in the time determina-
tions are at the most two minutes.
3.2. Comparison between the Mean Kinetics
within Each Procedure
The overlays of mean kinetics at different methanol con-
centrations from protocols A and B are showed in Fig-
ures 1 and 2, respectively. Mean kinetics at the single
methanol concentrations are also showed in these figures
to ensure clarity of details.
As expected, the interfering effect of methanol caused
the formation of an atypical peak on some reaction curves
(Figures 1(a)-(c), Figures 2(a)-(d)), producing an unusual
kinetic behaviour. In fact, when the F-C reaction was car-
ried out in the presence of methanol, fine solids formed
and precipitated in time (clouding and precipitation), out-
lining the left and the right sides of the peak, respectively.
The whole phenomenon appeared spectrophotometrically
unhomogeneous, showing high standard deviations
(Figures 1(a)-(c), Figures 2(a)-(d)). Moreover, it was
rather general with the exception of the kinetics carried
out in the presence of 2% and 4% methanol from proce-
dure A, where the phenomenon never appeared (Figures
1(d)-(e)), and in the presence of 2% methanol from pro-
cedure B, where only the beginning of the clouding was
possible to observe (Figure 2(e)).
On the basis of a comparative analysis among the
mean kinetics within each procedure we can claim that
both the appearance and the duration of peaks are corre-
lated with methanol concentration. In particular, the
peaks of mean kinetics within each procedure shift and
the phenomenon duration increases with decreasing
methanol concentration (Figures 1 and 2). The behav-
iours observed within each procedure can be ascribed to
the fact that, as the methanol concentration rises, solute
(i.e. inorganic salts or ionic species) solubility in reaction
mixtures decreases. Theoretically, alcohol affects the
dielectric constant of the medium, the inter-ionic attrac-
tion, and the solute-solvent interaction [15]. Thus, alco-
hol can reduce the solubility of inorganic salts decreasing
on the one hand the solvation of the ions and on the other
the dissociation of salts. In any case, when an alcohol
solvent is mixed with a salt water solution, the dielectric
property of the newly-formed mixed solvent decreases
and the solubility of the solute in the reaction mixture
correspondingly decreases [16] bringing about the pre-
cipitation phenomenon observed. Furthermore, it was
also reported that the presence of alcohol accelerated the
particle growth rate [17].
It is important to mention briefly that the precipitate
formation ensues from a two-step process. In the first
step, called nucleation, some tiny particles known as
nuclei are generated. In the second step, called growth,
the nuclei grow and the visible solid-phase particles are
formed. The rates of these two consecutive steps deter-
mine the particle size and its distribution [18]. Moreover,
it is worth recalling that, the supersaturation of reaction
mixture, the ability to dissolve more solids than that of
its equilibrium state, is the primary driving force for the
precipitate formation. Generally, a higher degree of su-
persaturation can generate a rapid nucleation and a slow
growth. On the contrary, a lower degree of supersatura-
tion promotes a slow nucleation and a fast growth [19]. It
is known that the control of supersaturation requires the
knowledge of all the variables affecting the formation of
fine solids, such as impurities, temperature, pH ect. The
effects of these parameters on F-C method in the pres-
ence of methanol are very interesting and still need to be
completely understood.
In this section and in following one, we discussed the
results about the effect from the initial carbonate concen-
tration on F-C method in the presence of an increasing
methanol concentration in the final reaction mixture.
In the context of this section, the difference observed can
be attributed to the fact that, as the methanol concentration
rises, the degree of supersaturation of solute in reaction
mixtures decreases due to the resulting reduction in
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Figure 1. Comparisons between mean kinetics at the different methanol concentrations within protocol A where a 5% carbonate
solution was used to reach the carbonate co ncentration equal to 4% in final mixture. Endnotes: the investigated re action mixtures,
each containing 8 mg/l ca ffeic acid, w ere monitored at 40 ˚C over a period of 2 h recording the absorbance data every two minutes.
The mean absorbance values and standard deviations of the kinetics carried out in the presence of 10%-8%-6%-4%-2% methanol
are shown in the graph inserts a-b-c-d-e, respectively. Instead, only the m ean ab sorb ance valu es reco rded ev ery 4 min utes a re shown
to ensure clarit y of details .
Figure 2. Comparisons between mean kinetics at the different methanol concentrations within protocol B where a 20% carbonate
solution was used to reach the carbonate concentration equal to 4% in fina l mixtu re. Endnot es listed as in Figure 1 .
Copyright © 2011 SciRes. AJAC
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medium dielectric property. All this makes the phenome-
non more and more advanced. This opinion is corrobo-
rated by the fact that a lower dielectric constant of mixed
solvent corresponds to a lower solubility of inorganic sol-
ute and, thus, a shorter induction period for nucleation as
well as higher solid particle growth kinetics [16].
Figures 1 and 2 show that the maximum absorbance
of mean kinetics increases as the methanol concentration
increases with the exception of the 10% methanol con-
centration from protocol B (Figure 2). In this case, the
precipitate has already formed during the reaction expo-
nential phase making the peak very small; furthermore,
during the 2 h period, the colour development is lower
and the mean absorbance values show high standard de-
viations (Figure 2(a)). Presumably, the precipitate for-
mation is so fast that reduces the availability of the ionic
species for the development of blue colour.
3.3. Comparison between the Mean Kinetics
from the Two Procedures
Mean kinetics obtained at the same methanol concentra-
tion from the two procedures are showed in Figure 3.
Moreover, the data obtained by analyzing the incre-
mental ratios from the two procedures are showed in
summary Table 1. In particular, the beginning and the
end of the phenomenon, as well as the time where the
peak maximum absorbance was reached, are reported.
For clarity’s sake, the duration of clouding, precipitation
and the whole phenomenon are also reported. As can be
seen in Table 1, the duration of the clouding is always
shorter than the precipitation one, thus producing asym-
metrical peaks (Figure 3).
The thorough comparative analysis of the kinetics al-
lowed us to make the following considerations. The
peaks of the kinetics in the presence of 10%-8%-6%
methanol from protocol A shift with respect to those
from protocol B (Figures 3(a)-(c) and Table 1). More-
over, in the whole experiment period, the mean kinetic
obtained in the presence of 10% methanol from protocol
B shows lower absorbance values than those from pro-
tocol A (Figure 3(a)), similarly to what was observed
among the methanol concentrations within procedure B.
Furthermore, the mean kinetics obtained both in the
presence of 10% and 8% methanol from protocol A show
a higher maximum absorbance value than that of the re-
spective mean kinetic from protocol B (Figures 3(a)-(b)),
while an opposite behaviour can be observed in the mean
kinetic obtained in the presence of 6% methanol (Figure
Figure 3. Comparisons between the mean kinetics from the
two procedures A (white circles) and B (grey circles). The
mean kinetics obtained at the final methanol concentrations
equal to 2%, 4%, 6%, 8% and 10% were reported in plots
(a), (b), (c), (d), (e), respectively. The mean absorbance val-
ues are shown over a 2 h period every 4 min.
Finally, as mentioned in section 3.2, the kinetics obtained
in the presence of 4% and 2% methanol show no peak when
using protocol A (Figures 3(d)-(e) and Table 1).
Table 1. Temporal features of the phenomenon due to the methanol effect on the F-C micro-method from procedures A and
B, where 5% and 20% carbonate as initial concentrations were used respectively.
Initial carbonate
concentration (%) 5 20
Final methanol
concentration (%) 10 8 6 4 2 10 8 6 4 2
Beginning 12 ± 218 ± 228 ± 2― ―12 ± 216 ± 2 20 ± 2 40 ± 58 ± 2
Peak maximum absorbance 34 48 92 ――28 38 48 88 n.d.
End 60 ± 286 ± 2n.d. ― ―42 ± 268 ± 2 82 ± 2 n.d. n.d.
Clouding duration 22 ± 230 ± 264 ± 2― ―16 ± 222 ± 2 28 ± 2 48 ± 2n.d.
Time (min)
Precipitation duration 26 ± 238 ± 2n.d. ― ―14 ± 230 ± 2 34 ± 2 n.d. n.d.
Phenomenon duration 48 ± 468 ± 4n.d. ― ―30 ± 452 ± 4 62 ± 4 n.d. n.d.
Instead, the phenomenon always appears when using
protocol B even if in the presence of 4% methanol the
end of the phenomenon cannot be determined (Tab le 1),
and in the presence of 2% methanol only the beginning
of the clouding can be observed in the 2 h period (Figure
3(e) and Table 1). On the basis of the results shown in
Figure 3 and Table 1 we can assert that the formation of
precipitating fine solids is delayed, slowed down and
even prevented when using procedure A instead of pro-
cedure B.
In our opinion, the differences observed could depend
on a different local supersaturation of the ionized species
in reaction mixtures probably due to two factors acting
simultaneously. The first is the different solvation of
carbonate ions in the solutions at 5% and 20%, used in
procedures A and B, respectively. The second is the dif-
ferent methanol concentration that the compared reaction
mixtures have at the moment of carbonate addition. In
fact, at that moment, methanol concentration, as well as
the F-C reagent concentration, in reaction mixtures from
procedure A is higher than those from procedure B.
The results support the idea that, before the reaction
equilibrium is reached, the different concentrations of the
species involved can give a different diffusion which, in
its turn, can cause a different local supersaturation in the
compared mixtures from the two procedures. Evidently
the local supersaturation is higher in procedure A than
procedure B triggering off the formation of many nuclei
growing more slowly. Consequently, the phenomenon o-
bserved during procedure A is delayed and slowed down
in comparison with that observed during procedure B
because smaller particles have formed more slowly.
Our opinion is proved to be right by preliminary SEM
analysis which highlights the different precipitate size
(Figure 4). Moreover, when using procedure A (5% car
bonate) the phenomenon does not appear at the 2% - 4%
methanol concentration because the reaction mixture
does not become supersaturated at these conditions than-
ks to the shielding effect of a high solvation degree of the
carbonate ions which predominates on the weak effect of
methanol due to its low concentration at these conditions.
On the contrary, and on the basis of what said above, the
evident difference observed between the mean kinetics
from both procedures in the presence of 10% methanol
can be due to the fact that the experiments conducted
using procedure B led to an early and fast formation of
bigger fine solids which precipitate more quickly than
those obtained when using procedure A.
Therefore, we can conclude that, although the same
carbonate amount is reached in final mixtures, the initial
carbonate concentration plays an important role in the
F-C reaction when this is carried out in the presence of
methanol since the generation of nuclei and their growth
is based on the local properties of mixture, rather than
the average properties.
3.4. Observation of Precipitate
After the 2 h period, by carefully observing the tested
mixtures in each cuvette, we remarked that the precipi-
tates were quite evidently visible to the naked eye when
carrying out the F-C reaction in the presence of 10% and
8% methanol in both procedures, even if the precipitate
from procedure B was slightly higher than that from
procedure A. Instead, when the F-C reaction was con-
ducted in the presence of 6% methanol using protocol B,
the precipitates were less visible and not always visible
using protocol A. In this latter case the peaks did not ap-
pear, either. In accordance with this behaviour, the pre-
cipitates were not always observed in the presence of 4%
and 2% methanol when using protocol B and never ob-
served when using protocol A.
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Figure 4. SEM images of precipitates obtained during F-C reaction carried out in the presence of 6% methanol from the
procedures A (a) and B (b).
On the basis of these observations, we can assert that
the yield of precipitation is strictly related to the stage of
precipitation which, in its turn, depends on supersatura-
tion degree. Nonetheless, the data of this study did not
allow us to clarify why the phenomenon is random in the
presence of 6% methanol when using protocol A as well
as 4% and 2% methanol when using protocol B.
The precipitates obtained in the presence of 6%
methanol from both procedures were also observed by
scanning electron microscope (SEM). SEM images, re-
ported in Figure 4, show that presumably the precipitate
consists both of crystalline and amorphous material. The
former is mostly represented by spindle-shaped crystals
which were bigger (about equal to and smaller than 10
m) in precipitate from procedure B than those from
procedure A (smaller than 5 m). The latter is mostly
represented by spherical particles with a mixture of some
ellipse-like particles. In any case, both the spherical par-
ticles and ellipse-like particles showed a mean bigger
diameter (about equal to and smaller than 10 m) in the
precipitate from procedure B than that from procedure A
(about equal to and smaller than 3 m). Moreover, some
of the observed particles were attached to each other for-
ming agglomerates.
3.5. Important Detail from Real Time Reaction
In accordance with the previous paper, our experiments
indicated that procedure A allows us to carry out without
any problem the F-C micro-method in the presence of
methanol up to the 4% concentration and, contrary to
what we expected, the precipitate never appeared at this
methanol concentration in the 2 h period. Surprisingly,
the results from procedure A also showed that a metha-
nol concentration equal to 6% can be reached in the final
mixture, as a new extreme condition, by making the real
time reading at 24 min. In fact, the mean absorbance
value at these conditions (Abs = 0.977 ± 0.019) was
similar to that found in the presence of 4% methanol at
20 min (Abs = 0.973 ± 0.008) corresponding to about
96% of blue colour development, taken as reference ab-
sorbance [7]. Moreover, by real time reading at 24 min
the F-C assay can still be carried out quickly, given that
neither filtration nor centrifugation techniques are nec-
essary because precipitates will appear later (Table 1).
3.6. Conclusions
The carbonate concentration used in the F-C microme-
thod, carried out in the presence of methanol, is an im-
portant parameter which should not be ignored since it
can modify the formation of precipitating fine solids due
to the interfering effect of methanol. The data presented
in this manuscript not only show in depth the methanol
effect on the F-C reactions but also highlight why the
initial carbonate concentration influences it. In particular,
the formation of particles is delayed and slowed down
with decreasing methanol concentration and can be fur-
ther delayed, slowed down and even prevented when a
carbonate solution at 5% instead of 20% is used. The cu-
rrent investigation, validating our hypothesis, allowed us
to conclude that the change of ionic species (reagent
and/or product) solubility in the F-C reaction is corre-
lated to concentrations both of methanol (concentration
in final mixture) and carbonate (initial concentration of
stock solution). Moreover, a local high supersaturation
can be formed when using procedure A instead of pro-
Copyright © 2011 SciRes. AJAC
cedure B resulting in a higher formation of nuclei grow-
ing more slowly.
Furthermore, we can assert that F-C assay can be car-
ried out in the presence of 6% methanol, as a new ex-
treme concentration, by real time reading the absorbance
at 24 min.
In our opinion, the real time monitoring of absorbance
can be a useful means to explore the effect of other pa-
rameters on precipitate formation caused by interference
from methanol in the F-C reaction.
The results of this study provide important information
for further investigations aimed at increasing the alcohol
concentration in the final F-C reaction mixture.
4. Acknowledgements
The authors wish to acknowledge Dr. Luca Medici and
Dr. Antonio Lettino for their scientific and technical as-
sistance in SEM analysis.
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