Raw material, different formulations of foods, and dietary supplements of
Thin layer chromatography (TLC) often represents first-choice technique for samples of plant origin as results can be easily visualized. This technique has been successfully employed for analysis of flavonoids and phenolic acids in wine, propolis, and different medicinal plants [
Determination of xanthines as the main constituents is time consuming titration [
All standards were purchased from Sigma Aldrich (Germany) and organic solutions from Kemika (Croatia). The sample of
Stock solutions of phenolic acids (chlorogenic, caffeic, ferulic, and
1.0 g of
0.5 g of
Total content of flavonoids was determined by Christ-Müller’s method, phenolic acids according to European Pharmacopoeia, and xanthines by the method suggested by International Office of Cocoa, Chocolate and Sugar Confectionery [
For the purpose of phenolic acids determination 0.2 g of
Aqueous solution of
Chloroform : methanol : formic acid in volume ratio 44.1 : 2.5 : 2.15 was used as mobile phase for aglycones, and ethyl acetate : formic acid : acetic acid : water in volume ratio 100 : 11: 11 : 26 was used for glycosides [
The results of TLC densitometry and spectrophotometric analysis were compared using Student’s
The total content of flavonoids determined by Christ-Müller’s method was 3.0 mg/g (RSD = 0.5%,
Identification of bioactive constituents was based on retention factor, absorption spectra
Identification parameters of polyphenolics and xanthines: retention factor, color of band under 366 nm, and spectra after spraying with aluminium chloride (not applicable for caffeine and theobromine).
Bioactive compound | TLC spectra |
Structure |
---|---|---|
Chlorogenic acid |
|
|
Ferulic acid |
|
|
Caffeic acid |
|
|
|
|
|
Naringenin |
|
|
Rutin |
|
|
Quercetin |
|
|
Kaempferol |
|
|
Caffeine |
|
|
Theobromine |
|
|
Identification of chlorogenic acid—overlapping spectra of standards and
Times of chromatogram development were 11.5, 24, and 12 min. for aglycones, glycosides, and xanthines, respectively. Out of eight polyphenolics analyzed rutin, and chlorogenic acid, as well as aglycones quercetin, kaempferol, and caffeic acid were identified and quantified. Both xanthines analyzed, caffeine, and theobromine, were determined (Figure
Results of quantification of individual flavonoids, phenolic acids, and xanthines.
Bioactive compound | Calibration curve | Fraction of flavonoids1 | Fraction of phenolic acids2 | Aqueous solution |
---|---|---|---|---|
(mg/g) | (mg/g) | (mg/g) | ||
Chlorogenic acid |
|
2.1 | 21.6 | 3.0 |
Caffeic acid |
|
1.5 | nd | nd |
Rutin |
|
5.2 | 6.8 | 2.3 |
Quercetin |
|
2.2 | nd | nd |
Kaempferol |
|
4.5 | nd | nd |
Caffeine |
|
na | na | 5.4 |
Theobromine |
|
na | na | 2.7 |
1Hydrolyzed fraction of flavonoids, 2nonhydrolyzed fraction of phenolic acids used for the spectrophotometric analysis according to the European pharmacopoeia, and nd: not detected, na: not applicable.
Chromatographic plate (a) and 3D chromatograms (b) of caffeine standard (six tracks from the left and nine from the right) and sample of
Although in the last few years HPLC has been a major method for the analysis of polyphenolics in
Although advanced techniques for the separation and quantification of individual polyphenols including LC-MS [
The results of TLC analysis concurred with the total content of methylxanthines: 5.4 mg/g for caffeine and 2.7 mg/g for theobromine, while total xanthines were 7.6 mg/g
Based on the obtained results, it can be concluded that TLC analysis is an appropriate technique for the analysis of individual polyphenols as well as xanthines. Content of individual methylxanthines corresponds to the spectrophotometric determined total xanthines.
As thin layer chromatography is readily available technique, it could be easily employed for the analysis of polyphenols and xanthines in raw material, different formulations of foods, and dietary supplements of
The authors declare no conflict of interests.
This work was financially supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Grants nos. 006-0061117-1237 and 006-0061117-1239).