The Influence of Initial Carbonate Concentration on the Folin-Ciocalteu Micro-Method for the Determination of Phenolics with Low Concentration in the Presence of Me-thanol: A Comparative Study of Real-Time Monitored Reactions

doi:10.4236/ajac.2011.27096

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American Journal of Anal yt ical Chemistry, 2011, 2, 840-848

doi:10.4236/ajac.2011.27095 Published Online November 2011 (http://www.SciRP.org/journal/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

E-mail: [email protected]

Received August 17, 2011; revised September 19, 2011; accepted October 8, 2011

Abstract

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-

841

N. CICCO ET AL.

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

time.

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.

N. CICCO ET AL.

842

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

N. CICCO ET AL.

843

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 .

N. CICCO ET AL.

844

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 .

N. CICCO ET AL.

845

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

3(c)).

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).

N. CICCO ET AL.

846

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.

847

N. CICCO ET AL.

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

Monitoring

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-

N. CICCO ET AL.

848

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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