Microchemical Journal 91 (2009) 107–110 Contents lists available at ScienceDirect Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m i c r o c A reproducible, rapid and inexpensive Folin–Ciocalteu micro-method in determining phenolics of plant methanol extracts Nunzia Cicco a,⁎, Maria T. Lanorte a, Margherita Paraggio a, Mariassunta Viggiano a, Vincenzo Lattanzio b a b Institute of Methodologies for Environmental Analysis, National Research Council 85050-Tito Scalo (PZ), Italy Department of Science Agro-Environmental, Chemistry and Plant Defense, University of Foggia, 71100- Foggia, Italy a r t i c l e i n f o Article history: Received 13 August 2008 Received in revised form 25 August 2008 Accepted 25 August 2008 Available online 1 September 2008 Keywords: Folin–Ciocalteu Micro-method Spectrophotometry Phenolics Plant methanol extracts a b s t r a c t A new combination among time, temperature, alkali and alcohol is described for the spectrophotometric determination of small concentrations of phenolics in methanol extracts from plant. It is a variation of the classical Folin–Ciocalteu (F–C) method, but the reaction conditions are optimized in order to eliminate methanol interferences in the assay. Alcohol concentration and reaction time limits have been evaluated as 4% methanol (v/v) and 20 min at 40 °C, using a 5% (w/v) sodium carbonate solution. This F–C micro-method is reproducible, quick, inexpensive and particularly helpful if it works with numerous samples or on a small scale, such as during the setting up of an experimental procedure of alcoholic extractions. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The quantitative determination of phenolic compounds by using Folin–Ciocalteu (F–C) reactive is a widespread method. It involves oxidation in alkaline solution of phenols by the yellow molybdotungstophosphoric heteropolyanion reagent and colorimetric measurement of the resultant molybdotungstophosphate blue [1,2]. These blue pigments have a maximum absorption depending on the qualitative and/or quantitative composition of phenolic mixtures besides on the pH of solutions, usually obtained by adding sodium carbonate. Although this method is routinely used in various laboratories, specific details of the method differ considerably. So, when the comparison of results from different laboratories becomes necessary, the comparability of the values obtained by each analyst is doubtful even if each relative value can be informative. The variations of the method, widely studied by Singleton and Rossi [1] and later by others [3,4], concern the initial and final concentrations of sodium carbonate, the sequence of reagent additions as well as the timing of these additions, and the time and temperature of reaction mixture incubation [1,5–10]. Wavelengths in the 700–760 nm range at which absorbance is determined and the final mixture volume in the 2–100 ml range are variable parameters too. Another detail that is important enough not to be ignored is the alcoholic concentration in the final mixture. In this context, Singleton et al. [11] assert that inclusion of solvents other than water in the ⁎ Corresponding author. Fax: +39 0971 427 222. E-mail address: cicco@imaa.cnr.it (N. Cicco). 0026-265X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2008.08.011 samples can sometimes interfere in the blue pigment formation, but an alcohol equivalent to 1 ml/100 ml of the final reaction mixture gives reproducible results by preparing standards and blanks in the same solutions of the samples. Moreover, the same authors affirm that, in order to obtain reproducible results, it is important to mix the sample and the F–C reagent under dilute conditions and to add a carbonate solution at the end. Therefore, the carbonate solution to be added in reaction mixtures may be relatively concentrated. In particular, they use a 20% (w/v) carbonate solution with samples/standards and the F–C reagent, of which one has previously been highly diluted, producing a 3% carbonate content in the final mixture. According to Singleton [1,11], most of the authors carry out the phenolic determination with a final concentration of alcohol not above 1% [2,5–9,12–14], adding a 20% carbonate solution [5,8,11–16]. Only few authors carry out the measurement with a higher final alcohol value [17,18] and use a lower initial carbonate, but similarly to Singleton, they accomplish the assay in the presence of carbonate lower than 3% in the final mixture at room temperature and for incubation time higher than 1 h. We encountered evident precipitations whenever the conditions reported in various F–C methods were not exactly and fully respected, in particular with regards to the alcohol concentration verifying that it results to be a parameter affecting the reproducibility in F–C assay. Therefore, depending on experimental needs, F–C methods may be inaccurate, inconvenient and/or time-consuming. Most of the protocols resulted particularly unsuitable for our needs because they required a concentration of alcohol in the final reaction mixture not higher than 1% and/or long incubation time. Drawbacks 108 N. Cicco et al. / Microchemical Journal 91 (2009) 107–110 were more evident especially if the samples were numerous and/or so little concentrated that they could not be diluted during the assay. In this paper, a rapid analytical procedure is described for spectrophotometric determination of phenolics. This procedure is based on the F–C method but used a new time–temperature–alkali– alcohol combination. It is suitable to determine phenolics quickly, also with low concentrations, from small amounts of plant methanol extracts, being it compatible with final concentrations of alcohol higher than 1%. 2. Materials and methods 2.1. Reagents and solutions Experiments were performed using F–C reagent from Merck (1.09001), sodium carbonate (S6139) and pure caffeic acid (C0626) from Sigma. Caffeic acid was chosen as standard phenol because of its abundance in our phenolic extracts. The detailed procedure for preparation of caffeic acid solutions was as follows. In a 100-ml volumetric flask 100 mg of caffeic acid, weighed using an analytical scale, were dissolved with 100% methanol. Then 1, 2, 3, 4, 5, 6, 7, 8, 9 ml of the above stock solution were diluted to 10 ml with distilled water to obtain different caffeic acid concentrations in methanol at 10, 20, 30, 40, 50, 60, 70, 80, 90% respectively. These solutions were stored at 4 °C for 1 week. 100 mg/l caffeic acid solutions for each methanol concentration were freshly prepared from above solutions by diluting them with methanol solutions at proper concentrations. Then, accurate serial dilutions of caffeic acid (80, 60, 40, 20, mg/l and 0-blank) were prepared to construct a calibration curve for each methanol concentration. Because of the interference of alcohol in the F–C method, all above described solutions must be prepared with great care checking the correspondence between the volumes of each methanol solution and the relative weights calculated on their relative density. Fig. 2. F–C reaction mixture in the presence of 8 mg/l caffeic acid, 4% carbonate, A–B–C– D methanol concentrations after assay at 40 °C for 20 min. A = 4% methanol, mixture appears completely clear; B = 6% methanol, fine solids begin to form; C = 8% methanol, evident precipitations appear; D = 10% methanol, mixture appears completely cloudy. cuvette by the spectrophotometer UV/visible ultrospec 4000 (Pharmacia). For calibration solutions and blank preparation, a methanolic solution at the same concentration of samples was used. 2.3. Statistical analysis All absorbance values, relative to the reaction mixtures tested, were evaluated statistically by analysis of the variance using one-way ANOVA (p ≤ 0.05). 3. Results and discussion 2.2. Analytical procedure for the proposed F–C micro-method 100 μl of properly diluted samples, calibration solutions or blank were pipetted into separate test tubes and 100 μl of F–C reagent were added to each. The mixture was mixed well and allowed to equilibrate. After exactly 2 min, 800 μl of a 5% (w/v) sodium carbonate solution were added. The mixture was swirled and put in a temperature bath at 40 °C for 20 min. Then, the tubes were rapidly cooled on the rocks and the colour generated was read at its maximum absorption, i.e.740 nm in the case of caffeic acid. The absorbance was measured in 1-cm Small amounts of phenolics in plant methanol extracts, obtained by solid phase extraction (SPE), were determined with some difficulties and/or low reproducibility by the different versions of the F–C method in particular with regards to alcohol concentration. It is well known that the combination between concentration of phenols, amount of Folin reactive, alkalinity and temperature in the F–C method causes variable times at which the maximum colour development is reached. In particular, this time is shortest with more Folin reactive, less phenolics, more alkali and warmer temperature but the complex blue produced is more stable with lower alkali and lower temperature [1,19,20]. We tested a new procedure of the F–C method making it faster, and compatible for alcohol concentrations higher than 1%, hence suitable to assay extracts with low phenolic content. In order to reduce reaction time of our F–C procedure, we carried out trials in the presence of 4% carbonate at 40 °C joining the effects of alkalinity and of temperature. These parameters were in accordance with Marigo [8], but different from Singleton [2] who asserts it would be advantageous to lower the carbonate content below 3% in the final mixture, if the temperature is higher than 20 °C. At the same time, to establish the maximum alcohol concentration compatible with our assay, we investigated the effect of different methanol concentrations (1–10%) in final mixtures using an initial carbonate concentration equal to 5% that is much lower than that used both by Singleton and Marigo (20%) but very similar to that used by Gao et al. (6%) [17,18]. 3.1. Determination of the maximum alcohol concentration Fig. 1. Effect of methanol on reaction kinetics accomplished at 40 °C in the presence of 4% sodium carbonate. The absorbance data, referred to mixtures containing 8 mg/l caffeic acid, represent means of 3 replicate × n (n = 4). The methanolic solutions (10–100%) with 80 mg/l caffeic acid, which is the upper limit concentration of the standard that can be N. Cicco et al. / Microchemical Journal 91 (2009) 107–110 Table 1 Caffeic acid determination by proposed method carried out in the presence of different methanol percentages in the final mixture Time (min) Absorbances (mean ± SD) 1% 2% 3% 4% 5% 6% 10 20 30 40 0.890 ± 0.029 0.971 ± 0.022 1.010 ± 0.030 1.043 ± 0.027 0.892 ± 0.025 0.970 ± 0.029 1.013 ± 0.024 1.040 ± 0.028 0.893 ± 0.029 0.970 ± 0.031 1.009 ± 0.031 1.041 ± 0.033 0.883 ± 0.025 0.977 ± 0.028 1.018 ± 0.030 1.061 ± 0.058 0.882 ± 0.021 0.968 ± 0.020 1.017 ± 0.024 1.083 ± 0.062 n.d. n.d. n.d. n.d. Absorbance data, referred to mixtures containing 8 mg/l caffeic acid, represent means of 3 replicate × n (n = 4). used in the assay, were analysed in a time interval of 40 min under the above mentioned conditions. For the sake of simplicity, Fig. 1 shows only reaction kinetics in the presence of 4% and 5% methanol in the final mixtures. No significant differences between the absorbances of each of the kinetics for methanol concentrations up to 4% were observed. A small increment of absorbance, due to slight suspensions forming, was observed after 30 min in the presence of 5% alcohol. We met great difficulties for alcohol concentrations higher than 5%. In fact, for these alcohol concentrations a loss of reproducibility already occurs at 20 min and it is caused by the presence of fine solids forming which are much more evident at increasing alcohol concentration and make spectrophotometric measurements impossible (Fig. 2). Table 1 shows the mean absorbances and the standard deviation values at 10, 20, 30 and 40 min, related to concentrations of methanol from 1% to 6% in the final mixture. In particular standard deviations up to 30 min are uniform and very small, while higher standard deviations are observed at 40 min starting from 4% methanol. We believe that precipitation events already begin at 4% methanol at 40 min even if they are not evident with the naked eye. The results of preliminary experiments show the possibility to reach, as extreme conditions, a methanol concentration equal to 5% and a maximum reaction time of 30 min. We chose to carry out the assay at 4% methanol in the final mixture because this concentration allows us to dilute the sample as little as possible, ruling out the extreme conditions. 3.2. Determination of reaction time In order to establish the lower reaction time at which developed blue colour can be considered maximum at above conditions, we 109 monitored the time–behaviour of absorbance curve related to a mixture containing 8 mg/l caffeic acid, 4% carbonate and 4% alcohol, 10% Folin, in the period of 8 h at 20 °C. At this temperature the final reaction mixture always appeared clear during all the incubation time. On the contrary, precipitates are observed at 40 °C in the period after 40 min making measurements impossible. Fig. 3 shows the comparison between the reaction kinetics of 8 h long at 20 °C and that of 30 min long at 40 °C. In the reaction kinetics at 20 °C, the mean absorbance at 4 h (Abs = 1.021) being about 95% of the mean absorbance measured at 8 h (Abs = 1.074), can represent a good approximation of the maximum absorbance value and be taken as reference absorbance for the kinetic at 40 °C. Consequently, the mean absorbances at 20 min (Abs = 0.977) and at 30 min (Abs = 1.018) measured at 40 °C represent the 95.7% and 99.7% of reference one respectively. These results suggest the possibility to accomplish the assay at 20 min as the lower limit of time. We consider this time long enough because the 96% of blue colour is already obtained. The choice to increase the temperature and the alkalinity, not going beyond the 40 °C and 4% carbonate in the final mixture, respects fully the general principles on reactions involved in the F–C method. In fact, the value of temperature and alkalinity reached in our final mixture allows us to obtain a more rapid phenol oxidization. At the same time, these parameters are not so high to compromise the stability of the yellow reactive or of the blue complex forming in a time up to 40 min. 3.3. Important details of proposed analytical procedure On the basis of results obtained, we recommend not only to dilute the sample until reaching a maximum methanol concentration of 5% in the final reaction mixture, but also initially to use a 5% carbonate volume equal to 800 μl, corresponding to 80% of the final mixture volume. In our opinion, this aspect of the procedure allows us to minimize the possible interference from alcohol on reactions involved in the F–C method and consequently affords us the possibility to work at final alcohol concentrations up to 5%. A similar F–C procedure using a concentration of carbonate lower than 20% in the presence of an alcohol concentration higher than Singleton's has already been met in literature [17,18] but the F–C assay was accomplished in the presence of carbonate lower than 3%, at room temperature and for longer incubation time. Therefore, the peculiarity of our quick F–C procedure allows us to determine phenolics from plant methanol extracts at low concentration Fig. 3. Comparison between the reaction kinetics related to mixtures containing 8 mg/l caffeic acid, 4% carbonate and 4% alcohol, in the periods of 8 h and 30 min at 20 °C and at 40 °C respectively. The absorbance data represent means of 3 replicate × n (n = 3). 110 N. Cicco et al. / Microchemical Journal 91 (2009) 107–110 with a high accuracy because alcohol, up to 5% in the final mixture, does not cause interferences and does not become a source of decreased reproducibility. Finally, with respect to the final volume of the reaction mixture, we reached a volume equal to 1 ml resulting smaller than that used in literature. In fact, some authors carry out the F–C assay in a volume equivalent to 10 ml [8,9,13] or higher [1,2,6,7,10,12,16]. Only few authors accomplish the F–C assay in a lower final volume but always not below 1.5 ml, either by simply reducing the volumes reported in Singleton method [14] or changing the parameters involved in the procedure [17,18] too. Furthermore, with respect to the sequence of reagent addition, according to Singleton and Rossi [1] but differently from other authors [21,22], in all our assays we added sodium carbonate after the F–C reactive because the colorimetric assay is more sensitive. 4. Conclusions The F–C procedure presented here is advantageous for several reasons. It is particularly effective if we work with numerous samples and on a small scale as during the setting up of an experimental procedure of alcoholic extraction from which small volumes of extracts with low concentrations can be obtained. Moreover, it not only uses the minimum volume of reactive and reduces waste, but it is fast enough without compromising the assay accuracy and reproducibility. 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