This paper describes the application of HPLC and CZE to analyze flavonoids in the leaves of
Flavonoids are a heterogeneous group of polyphenols (about 4000 substances) present in all plants and responsible for their color, growth, development, and immunity [
Due to its robustness, sensitivity, and versatility, HPLC-UV/PAD (high performance liquid chromatography-ultraviolet detection using a photodiode array detector) is the technique of choice for the analysis of flavonoids and other phenolic compounds in natural products [
This work compares the HPLC and CZE techniques applied in the analysis of flavonoids contained in these two
Leaves of
Rutin, quercetin, and kaempferol standards were obtained from Sigma (St. Louis, MO, USA). HPLC-grade acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from Mallinckrodt (Paris, Kentucky, USA). Analytical grade methanol (MeOH) and ethyl acetate (EtOAc) were purchased from Mallinckrodt (Xalostoc, State of Mexico, Mexico). Analytical grade chloroform (CHCl3) was purchased from Merck (Rio de Janeiro, Brazil). TLC plates of silica gel 60, without fluorescent indicator, were purchased from Merck (Darmstadt, Germany). Analytical grade monobasic potassium phosphate (KH2PO4) and sodium tetraborate decahydrate (NaB4O7·10 H2O) were purchased from Reagen (Rio de Janeiro, Brazil). Analytical grade formic acid (HCOOH), phosphoric acid (H3PO4), hydrochloric acid (HCl), sodium hydroxide (NaOH), and polyethylene-glycol (PEG 400) were obtained from Synth (São Paulo, Brazil). Diphenylboric acid 2-aminoethylester (C14H16BNO) was purchased from Sigma (St. Louis, MO, USA). Water was purified in a Millipore Milli-Q Water Purification System (Eschborn, Germany). Hydrophobic Fluoropore (HF-PTFE) membranes (0.5
1.0 g of the
0.01 g of each flavonol standard (rutin, quercetin, or kaempferol) was dissolved separately in 10 mL of MeOH. An aliquot of 0.1 mL of each stock solution was diluted to 10 mL with MeOH to obtain a stock solution containing the three flavonols; this stock solution was utilized in the HPLC and CZE analyses.
Analyses were carried out on silica gel 60 aluminum sheets precoated with EtOAc/HCOOH/H2O (6 : 1 : 1 v/v). After developing the plates, the solvent was dried and the flavonoids were visualized with diphenylboric acid 2-aminoethylester-PEG 400 under UV at
The CZE analysis was performed in an HP3D Capillary Electrophoresis System (Hewlett Packard, Waldbronn, Germany) equipped with a photodiode array (Hewlett Packard) and an HP Chem Station data processing system. Separations were performed using an uncoated fused silica capillary tube (Polymicro Technologies, Phoenix, AZ, USA) with a total length of 64.5 cm, effective length of 56.0 cm, and i.d. of 50.0
This analysis was performed in a modular LC System (Shimadzu, Kyoto, Japan) consisting of two LC-10 AD pumps; a CTO-10A column oven; an SPD-M10A variable wavelength diode array detector; the LC-10 Workstation Class data processing system. Supelcosil columns (Supelco, Bellefonte, PA, USA) with stationary phase C-18 and C-8 columns (
Prior to the HPLC analysis, the
Optimization of the chromatographic conditions showed that the C-18 and C-8 columns were highly efficient in the separation of flavonoids from
The HPLC-UV/PAD analysis led to the detection of two flavonoids in
HPLC/DAD-UV (
The flavonoid peaks can be identified by their characteristic UV/PAD spectral pattern with two bands, Band I,
HPLC/DAD-UV (
In the chromatogram of
HPLC/DAD-UV (
Peak 8 was identified as rutin by direct comparison (retention time and UV-DAD spectra) with an authentic commercial standard (Figure
HPLC/DAD-UV (
HPLC/DAD-UV (
Figures
CZE/DAD-UV electropherogram of
CZE/DAD-UV electropherogram of
The CZE separation was optimized based on the parameters of pH, buffer concentration, and the effect of modifier. An important parameter is pH, which changes the electroosmotic flow (EOF) and affects the degree of ionization of the solutes. The electrophoretic mobility (
Effect of pH variation on the values of migration time (
pH | ||||||
---|---|---|---|---|---|---|
8.0 | 9.625 | 8.983 | 12.399 | 1.671 | 1.433 | 2.372 |
8.5 | 9.512 | 9.597 | 12.613 | 1.651 | 1.679 | 2.430 |
9.0 | 9.347 | 10.587 | 12.869 | 1.850 | 2.228 | 2.733 |
9.3 | 10.040 | 12.537 | 14.576 | 1.837 | 2.435 | 2.772 |
9.5 | 10.248 | 13.742 | 15.375 | 1.880 | 2.628 | 2.861 |
10.0 | 11.475 | 18.693 | 19.741 | 1.940 | 2.954 | 3.040 |
Figure
Resolution (Rs) between the peaks corresponding to the quercetin and kaempferol derivatives found in
Concentration of tetraborate/phosphate (mmol/L) | Rs1,2 |
---|---|
10/5 | Coelution |
30/5 | 5.506 |
50/5 | 6.320 |
30/25 | 5.941 |
30/50 | 6.311 |
Effect of pH on electrophoretic mobility of flavonols standards (rutin, quercetin, kaempferol). Conditions: buffer 30 mmol/L tetraborate, 50 mmol/L phosphate; for other electrophoretic conditions, see experimental part.
The results showed that the decrease in tetraborate concentration diminished the resolution in the separation of the flavonol glycosides due to the minor presence of tetraborate complexes at this concentration. On the other hand, increasing the tetraborate and phosphate concentrations led to a decrease in EOF and an increase in migration time due to the higher viscosity of the buffer. The resolution was calculated using the peaks of kaempferol and quercetin derivatives (major flavonoids), and the best results were achieved with 50/50 mmol/L tetraborate/phosphate. However, 30/50 mmol/L tetraborate/phosphate showed better separation if one also considers the minor flavonoids, so the latter proportion was chosen as the optimum condition for both
Figures
Effect of percentage organic modifier on efficiency (N) in the CZE analysis of
Effect of percentage organic modifier on efficiency (N) in the CZE analysis of
The HPLC and CZE techniques can both be used in the analysis of flavonoids in
The authors thank Dr. Ana Maria Soares Pereira for kindly supplying plant material, Professor Dr. Emanuel Carrilho for the discussions about CZE, and FAPESP (98/04334-9, 00/11645-2, 02/00493-2, and 06/59457-6), CNPq, and CAPES for granting fellowships and financial support.