3.1 Optimum electrolyte composition and CE conditions
During method development and optimization, the chromatographic resolution statistic equation (CRS) was used. This is a mathematical function developed originally for chromatographic separations 35; the CRS tends to a minimum value for chromatograms having well resolved and uniformly spaced peaks. It has been used in the CE area for optimizing the separation of fatty acids 36 and food dyes 37.
STB-based buffers, MeOH as organic modifier, SDS or Brij35 as surfactants, time/pressure of injection, voltage, and temperature were investigated. So that the effects of various factors could be simultaneously investigated, including interaction of factors, a factorial design was employed rather than the one-factor-at-a-time approach.
STB as the electrolyte was assessed from 10 to 30 mmol/L. The lower level was established considering the need to maintain the pH in all the analyses. Very low levels of STB do not provide good buffering capacity. The upper level was limited by the electrical current. High amounts of STB combined with high levels of SDS may result in a high current, and temperature gradients can occur in the capillary due to Joule heating.
MeOH as organic modifier was evaluated from 0 to 10%. One of the first effects is a decrease in the electrical current, reducing Joule heating and peak band broadening. The second effect is increasing the migration time and the resolution of the peaks. Modification of the pKa of the analytes can occur with MeOH, thereby increasing solubilization.
The third variable was SDS, evaluated from 0 to 10 mmol/L. The formation of micelles was the main objective in selecting this variable because each compound can partition to the micelle core, thereby increasing selectivity. Brij35 from 0 to 5 mmol/L was also included because the presence of both SDS and Brij in the electrolyte results in a mixed micelle system constituted by anionic monomers (SDS) and neutral monomers (Brij35). The mixed micelle might provide better selectivity.
Time of injection from 1 to 3 s at 10 mbar, operation voltage from 10 to 25 kV, and temperature from 25 to 35°C were also evaluated.
Optimization of the CE method was initially carried out, using a factorial design, for the selection of the factors with direct influence on the separation. After selection of these factors, they were optimized by a response surface methodology. Table 1 shows the studied variables for the factorial design as well as the low (−1) and high (+1) levels.
Table 1. 27-3 Factorial design variable levels
|Coded levels||STB (mmol/L)||MeOH (% v/v)||SDS (mmol/L)||Brij 35 (mmol/L)||Injection time (s/mbar)||Voltage (kV)||Temp (°C)|
The results of the 27-3 fractionary factorial design are shown in Table 2, revealing that the effects of the Brij35 and SDS levels were not significant. Likewise, the time of injection did not have any significant effect during the studied interval and the high level was chosen in order to obtain better sensitivity. The temperature was kept at a low level to avoid bubbles that could be formed by the evaporation of the organic solvent. The most significant effects came from the variables STB, MeOH, and their interaction. These variables were therefore studied further through the response surface methodology, keeping the voltage at 25 kV, injection for 3 s at 10 mbar, and the temperature at 25°C, without SDS and Brij35.
Table 2. Principal effects obtained from the factorial design 27-3
According to Table 3, the best condition (lowest CRS value) was achieved with 20% MeOH and 10 mmol/L STB. However, these conditions did not give good separation of the phenolic compounds of wine. Thus, response surface analysis, using the reciprocal of the CRS value, was used to obtain the optimum separation condition: 20% MeOH, 17 mmol/L STB, 25 kV, 3 s inj. 10 mbar at 25°C (Fig. 1).
Figure 1. Response surface diagram for electrolyte composition and CE conditions. Optimum conditions: 20% MeOH, 17 mmol/L STB, 25 kV, 3 s injection 10 mbar, 25°C. STB, sodium tetraborate; CRS, chromatographic resolution statistic.
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Table 3. Response function results of the response surface analysis
|Experiment||% MeOH v/v||STB (mmol/L)||CRS|
3.2 Method performance
Linearity was evaluated by the injection of five standard solutions of each compound at concentrations varying from 2.5 to 220 mg/L. LOD and LOQ were calculated as the solution concentrations that gave signal-to-noise ratios of 3 and 10, respectively. Peak and migration time precision was evaluated by ten consecutive injections of the standard solutions. Recovery was studied at three levels, each level injected in triplicate.
Table 4 shows the performance characteristics of the proposed method. At the concentration range tested (2.5–220 mg/L), the method presented good R2 values (from 0.994 to 0.999). The LOQ varied from 0.4 to 0.8 mg/L and the LOD from 0.1 to 0.3 mg/L. Instrumental precision was shown by RSDs of ten consecutive injections of standard solutions being lower than 2% for both peak area and migration time. Recoveries varied from 97% (myricetin and quercetin) to 102% (rutin). The method is therefore considered suitable for the analysis of phenolic compounds in wine.
Table 4. Performance characteristics of the CE method developed
|Compound||Concentration range (mg/L)a)||Intercept||Slope||R2a)||LOQa) (mg/L)||LODa) (mg/L)||Recovery (%)b)||Repeatability (%RSD)c)|
| || || || || || || || ||Peak area||Migration time|
3.3 Polyphenols of Brazilian wines
Obtained under the optimum conditions established, Fig. 2 shows the separation of the ten phenolic compounds used as standards. Figures 3 and 4 present the typical electropherograms of white and red wines, demonstrating the now well-known much greater phenolic concentrations in red wine. Table 5 shows the concentrations of the ten phenolic compounds determined in 23 inexpensive Brazilian wines. The wide variations in the phenolic levels indicate that better quality control for these wines is warranted.
Figure 2. Electropherogram of a mixture of phenolic compound standards. Peak identification: 1, resveratrol; 2, catechin; 3, rutin; 4, syringic acid; 5, kaempferol; 6, p-coumaric acid; 7, myricetin; 8, quercetin; 9, caffeic acid; 10, gallic acid. CE conditions are described in the text.
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Figure 3. Electropherogram of the phenolic compounds in red wine. Peak identification: 1, resveratrol; 2, catechin; 3, rutin; 4, syringic acid; 5, kaempferol; 6, p-coumaric acid; 7, myricetin; 8, quercetin; 9, caffeic acid; 10, gallic acid. CE conditions are described in the text.
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Figure 4. Electropherogram of the phenolic compounds in white wine. Peak identification: 1, resveratrol; 2, catechin; 3, rutin; 4, syringic acid; 6, p-coumaric acid; 8, quercetin; 9, caffeic acid; 10, gallic acid. CE conditions are described in the text.
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Table 5. Phenolic compound concentrations (mg/L)a) in Brazilian wines obtained by the CE method developed
|Sample||Resveratrol||Catechin||Rutin||Syringic acid||Kaempferol||Coumaric acid||Myricetin||Quercetin||Caffeic acid||Gallic acid|
|Cabernet Sauvignon red 2000||3.1±0.1||122.6±0.1||12.5±0.2||4.9±0.1||14.7±0.8||nd||12.3±0.1||11.7±0.1||11.7±0.1||29.1±0.1|
|Cabernet Sauvignon red 2001||3.5±0.3||44.7±0.1||23.6±0.4||4.4±0.1||17.4±0.6||nd||12.8±0.1||1.0b)||17.1±2.0||31.0±3.0|
|Cabernet Sauvignon red||1.4±0.2||111.2±0.1||23.5±0.5||4.5±0.3||16.1±0.5||12.7±0.1||12.3±0.1||14.2±0.1||15.9±0.3||15.0±1.0|
|Cabernet Sauvignon red||3.1±0.1||82.3±0.2||25.5±0.2||4.9±0.1||18.3±0.4||nd||12.3±0.1||11.7±0.1||11.7±0.1||29.1±0.1|
|Cabernet Franc red 2000||2.3±0.1||44.3±0.2||32.4±0.4||4.5±0.1||19.2±0.5||13.6±0.1||12.1±0.2||15.6±0.4||17.9±0.3||30.8±1.0|
|Cabernet Franc red 2001||3.4±0.1||132.1±0.1||24.5±0.5||4.4±0.1||12.1±0.5||12.9±0.1||11.9±0.1||14.8±0.3||16.4±0.7||37.6±0.1|
|Gammay red 2002||3.7±0.1||81.5±0.1||23.8±0.7||nd||12.9±0.4||nd||11.1±0.2||10.6±0.3b)||14.0±0.4||45.0±0.4|
|Gammay red 2003||1.0±0.1b)||117.8±0.1||24.8±0.5||4.5±0.3||15.4±0.7||12.5±0.2||11.7±0.3||15.1±0.5||15.2±1.6||56.4±2.1|
|Merlot red 2001||2.0±0.1||72.1±0.1||21.4±0.6||nd||nd||nd||11.2±0.3||11.5±0.2||17.7±0.1||68.7±0.2|
|Shiraz red 2002||1.4±0.2||72.9±0.2||21.2±0.6||nd||12.5±0.6||10.0±1.0||12.0±0.1||15.9±0.2||17.0±0.1||57.7±0.1|
|Chardonnay white 2002||nd||12.4±0.4||8.9±0.2||nd||nd||nd||1.6±0.1||1.7±0.4b)||1.8±0.3||1.6±0.4|
|Riesling white 2000||nd||13.6±0.4||nd||nd||nd||nd||nd||nd||2.2±0.1||2.2±0.2|
|Riesling white 2001||0.9±0.2b)||15.7±0.3||3.2±0.1||1.3±0.1||nd||2.1±0.3||nd||nd||4.4±1.5||2.5±1.2|
|Riesling white 2002||nd||18.9±0.4||nd||nd||nd||nd||nd||nd||3.4±0.3||2.5±0.5|
|Sauvignon Blanc white 2002||nd||23.4±0.4||nd||nd||nd||nd||nd||nd||2.9±0.2||2.9±0.2|
|Ugni Blanc white 2002||nd||21.3±0.2||nd||nd||nd||nd||nd||nd||2.2±0.4||2.8±0.2|
In the comparison that follows, the blended red wine is not included. Gallic (15.0–69.9 mg/L for red wine; 1.6–2.9 mg/L for white wine) and caffeic (11.7–17.9 mg/mL for red wine; 1.8–4.4 mg/L for white wine) acids were the only phenolic compounds found in all the wines. German and Walzem 10 put together published data on phenolic compounds of wine and the ranges were 26–320 mg/L gallic acid and 3–18 mg/L caffeic acid for red wines. The Brazilian wines, therefore, are in the lower end for gallic acid but with caffeic acid in the upper end of these ranges.
Myricetin was found in all red wines (11.1–12.8 mg/L) and one white wine (1.6 mg/L). Quercetin was also encountered in all red wines (1.0–15.9 mg/L) and in one white wine (1.7 mg/L). According to German and Walzem, myricetin (2–45 mg/L) and quercetin (5–53) were found only in red wines, the Brazilian values falling within these ranges.
Syringic acid was detected in 7 (4.4–4.9 mg/L) out of 11 red wines and in 1 white wine (1.3 mg/L). The range reported by German and Walzem was 4.2–5.9 mg/L for red wine, in agreement with the data of the present work.
Coumaric acid was greater in blended wines (14.6–19.9); the range in five red wines was 10.0–13.6 mg/L. These ranges agree with that of German and Walzem (7.5–22 mg/L for red wine), but this acid was not detected in six red wines.
The concentration of one of the most important phenolic compounds in wine, resveratrol, ranged from 1.0 to 3.7 mg/L in red wine. The range in blended wines was 0.4–1.4 mg/L; in white wine, only one had resveratrol at a low concentration (0.9 mg/L). Souto et al. 28 found 0.82 to 5.75 mg/L in eight types of Brazilian red wines, in agreement with the values obtained in the present work, slightly higher than the range of German and Walzem (0.1–2.3 mg/L for red wine).
For catechin, the red wines had a range of 44.3–183.2 mg/L, while the white wines had 13.6–23.4 mg/L. The ranges in German and Walsem were 27–191 mg/L for the former and 3–35 mg/L for the latter. Rutin had a range of 12.5–32.4 mg/L in Brazilian red wines, higher than the range of German and Walzem of 0.5–10.8 mg/L. This compound was not detected in white wine by German and Walzem; it was found in two Brazilian white wines at 8.9 and 3.2 mg/L. Kaempferol had only one value in German and Walzen's table for red wine: 18 mg/L, which is within the range encountered in the present study of 12.1–19.2 mg/L for nine red wines, not being found in two red wines. In both work, this compound was not detected in white wine.