Evaluation

Several characteristic parameters can be interpreted as the main evidence that an imprinting effect has been achieved (see Fig. 2 and Table 2). First, the retention time of quercetin in the column packed with the quercetin imprinted polymer is approx 10 times higher compared to the column packed with the control polymer. The imprinted column shows some cross-selectivity for structurally related compounds, as shown in Table 2. Nevertheless, the strongest difference in retention time between the imprinted and the control column occurs for the imprint molecule quercetin (retention index [RI] value of 1). According to Table 2, three analytes (Rutin, [+] Catechin, and C-fla) have low crossreactivity with the generated antiquercetin recognition cavities, whereas morin, with an RI value of 0.7 (see Note 10), shows considerable cross-reactivity owing to the closest structural similarities of the molecular structure to the imprint molecule quercetin. Second, the large difference in capacity factors for quercetin when comparing imprinted and control polymer (k'MIP = 10, k'CTL = 1.4 in MeCN:H2O:HAc [80:10:10]) indicated a strong imprinting effect. Third, the considerably pronounced tailing of the flavonoid peaks in the imprinted column is an indication of a heterogeneous binding site distribution with—more or less—specific and nonspecific sites distributed within the poly-

Quercetin imprinted polymer

Control polymer

Quercetin imprinted polymer

30 40

Time (min)

30 40

Time (min)

Control polymer

400

i

i 1 i

l

r

j

1

350

-

300

-

Morin,

250

-

Quercetin

-

200

-

-

O

-

100

-

50

-

0

i . i

i

i

i

i

0

10 20

30

40

50

Time (min)

Fig. 2. Chromatograms for (A) imprinted polymer and (B) control polymer with acetone, morin, and quercetin (4-VP/EDMA copolymer) in MeCN:H2O:HAc (80:10:10 [v/v/v]) as mobile phase (flow rate: 1 mL/min).

Table 1

Selected Flavonoid Compounds Used in HPLC Experiments

Table 1

Selected Flavonoid Compounds Used in HPLC Experiments

Name

Quercetin

Morin

C-Fla

Class

flavonol

flavonol

isoflavone

Formula

C15H10O7

C15H10O7

C19H16O7

CAS#

117-39-5

480-16-0

15485-76-4

Structure

Name

(+)-Catechin

Rutin

Class

flavanol

flavonol glycoside

Formula

C15H14O6

C27H30O16

CAS#

154-23-4

153-18-4

Structure

Table 2

Results for Capacity Factors, Separation Factors,

and RIs for Imprinted and Nonimprinted 4-VP/EDMA Copolymer

in MeCN:H2O:HAc (80:10:10 [v/v/v]) as Mobile Phase3

fCTL kCTL

¿MIP

kMIP aCTL

aMIP

RI value

Acetone

3.5

3.6

- -

-

-

Quercetin

8.4 1.4

39.5

10.0 1.0

1.0

1.0

Morin

8.4 1.4

27.8

6.8 1.0

1.5

0.7

(+) Catechin

4.9 0.4

5.9

0.7 3.5

15.2

0.2

Rutin

4.3 0.2

4.9

0.4 6.3

27.9

0.2

C-fla

4.5 0.3

4.7

0.3 5.0

33.5

0.2

"Flow rate: 1 mL/min. Bold numbers indicate values for the tempated analyte.

"Flow rate: 1 mL/min. Bold numbers indicate values for the tempated analyte.

mer (28). The print molecule interacts differently with these binding sites; it is retained for a longer time in the column owing to these interactions and, consequently, produces a broader peak with pronounced tailing. These results emphasize that HPLC columns packed with imprinted polymer particles are suitable for the separation of structurally closely related analytes based on biomimetic recognition mechanisms within a synthetic receptor matrix.

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