3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
Results
Since luteolin, quercetin, apigenin and kaempferol have
very similar structures (Fig. 1.), the main point of view of
this study was to find optimal analytical conditions for their
efficient separation.
Several single columns with different column lengths
and different sorbent parameters were tested. The gradient
of water/acetonitrile mixture was used as mobile phase. The
replacement of acetonitrile with methanol as mobile phase
component was also investigated, but the separations didn’t
show satisfactory results. After modification of mobile phase
compositions by formic acid at pH = 2.3, significant peak
shape improvement of the observed compounds was noticed.
In all experiments, the flow rate of mobile phase was
0.5 ml min –1 , only when Xterra C18 column was tested, the
flow rate was set to 0.3 ml min –1 because of the high column
backpressure. The experiments were performed with flavonoid
standard solution (c = 30 µg ml –1 ). Injected amount of
analyte was 20 µl.
Fig. 1. Chemical structures of analyzed flavonoids
The comparison of column efficiency for separation of
the above compounds is shown in Fig. 2. First, two hybrid
C18 columns with the same column parameters were compared:
(a) 3.0 mm i.d., XBridge 150 mm long column, 3.5 µm
particle size, (b) 3.0 mm i.d., Xterra 150 mm long column,
3.5 µm particle size. Both separations showed only three
peak. In separation (a) very nice separation of apigenin and
kaempferol was observed, while in separation (b) the separation
of apigenin and kaempferol was slightly poor. However,
the column performance of both columns was too poor to
separate luteolin and quercetin, the peak coelution was observed.
nevertheless, neither the utilization of longer column,
(c) 4.6 mm i.d., Supelcosil C-18 250 mm long column, 5 µm
particle size, didn’t show the improvement of luteolin and
quercetin separation and also the separation of kaempferol
and apigenin wasn’t very acceptable. Further, (d) 3.0 mm
i.d., Atlantis T3 C18 150 mm long column, 3.0 µm particle
size was tested. The column separated not only apigenin
and kaempferol but also luteolin and quercetin. However,
although the column proved to separate single flavonoids, the
peak shapes didn’t match our ideas. The efficient separation
of observed compounds was achieved by (e) 4.6 mm i.d.,
Symmetry C18 150 mm long column 5 µm stationary phase.
The kaempferol and apigenin were separated to baseline and
separation of luteolin and quercetin was sufficient for succesful
determination and quantification of both compounds
present in the sample.
s652
Fig. 2. hPLC chromatogram of comparison column separation
of flavonoid standards. (1) quercetin, (2) luteolin, (3) apigenin,
(4) kaempferol. (a) xbridge C18 column (3.0 × 150 mm length),
(b) xterra C18 column (3.0 × 150 mm length), (c) Supelcosil
C18 column (4.6 × 250 mm length), (d) Atlantis T3 C18 column
(3.0 × 150 mm length), (e) Symmetry C18 column (4.6 × 150 mm
length). For chromatographic conditions see the experimental
part
Conclusion
The data presented in this report indicate that there
are wide variations in the effectiveness of different reversed-phase
HPLC columns when used to analyse flavonoids.
High efficiency separation was obtained with Symmetry C18
column. Gradient elution with acetonitrile in water adjusted
to pH 2.3 by formic acid facilitated the separation of luteolin,
quercetin, kaempferol and apigenin. In the future, this procedure
will be used for quantitative analysis of Stevia flavono-