Supplementary Information
1
2
3
A miniaturised molecularly imprinted polymer extraction
4
method for the gas chromatographic analysis of flavonoids
5
Journal of Separation Science
6
7
8
9
Yada Nolvachai1, Chadin Kulsing2, Reinhard I. Boysen2, Milton T.W. Hearn2
10
and Philip J. Marriott1*
11
1
12
13
Australian Centre for Research on Separation Science, School of Chemistry, Monash
University, Wellington Road, Clayton, Melbourne, VIC 3800, Australia.
14
15
16
2
School of Chemistry, Monash University, Wellington Road, Clayton, Melbourne, VIC 3800,
Australia.
17
18
1
19
Experimental selection of the most appropriate monomer for MIP construction
20
A catechin, morin and quercetin standard mixture was dissolved in 50% ethanol/water (50 mg
21
L-1 each). 100 L of mixture (corresponding to 50 × 10-4 mg) was loaded into separate MIP-
22
or NIP-modified micropipette tips. The loaded solution was pumped at ≈4 atm through the
23
modified tip to dryness (no residual solvent) by using the extraction kit. An aliquot (100 µL)
24
of the loaded solution could be collected within 10-20 min. This fraction is called the “flow
25
through fraction”. Absolute ethanol (100 µL) was then applied to wash further un-retained
26
compounds out of the tip (at 4 atm; the washed fraction was not collected in this step).
27
Subsequently, 10% acetic acid in THF (200 µL) was applied to the tip in order to elute the
28
compounds that had been captured by the monolithic MIP (or NIP) in the micropipette tip.
29
This collected fraction is called the “eluted fraction”.
30
31
Results and Discussion
32
To investigate extraction performance, 4VP and MA modified micropipette tips were tested
33
with a standard solution of loading concentration (50 mg L-1), a concentration presumed to be
34
greater than the flavonoid content in the black tea sample (refer to Section 3.3 in the
35
manuscript). MIP- and NIP-modified micropipette tips were used for extraction of three
36
flavonoid standards, to test capacity Table S1. The flavonoid content in the flow through
37
fraction was significantly lower for MIP compared with NIP polymers prepared with MA.
38
This indicates a selective binding for all compounds by the MIP, though to a different extent.
39
Compared to the result of a standard mixture without extraction (i.e. direct analysis), catechin,
40
morin and quercetin were captured by the MA-MIP (43, 55 and 63%, respectively). These
41
percentages are relatively low due to the washing process with absolute ethanol prior to the
42
Eluted fraction analysis (see the experimental part above). Quercetin was the most strongly
43
captured analyte, indicating that the quercetin imprinted MIP has a preference for this
44
molecule vis-à-vis its closely related analogues.
45
46
Capacities of MA-MIP and MA-NIP were compared (including both specific and non-
47
specific) for quercetin; the MA-MIP value was 0.95 mg g-1, and the MA-NIP was 0.54 mg g-1
48
with the higher value for the MA-MIP attributed to the imprinting effect. The capacities for
49
all three flavonoids were insignificantly different and higher in all cases for the MA-MIP than
50
the MA-NIP. In contrast to MA-MIPs, the flow through fraction was similar for the MIP and
51
NIP materials prepared with 4VP, except for morin which was the dominant analyte captured
2
52
by the 4VP-MIP. As the difference between MIP and NIP binding for morin was very low,
53
this points to non-specific binding.
54
55
Data revealed a lower imprinting effect for the 4VP-MIP compared to that of MA-MIP, as
56
well as significantly lower binding for both NIPs compared to their corresponding MIPs.
57
Subtraction of the binding capacity of MA-NIP from that obtained for the MA-MIP revealed
58
MA-MIP imprint specific binding capacities of 0.18, 0.21 and 0.41 mg g-1 for catechin, morin
59
and quercetin, respectively, whilst the corresponding values for the 4VP-MIP were 0.05, 0.10
60
and 0.06 mg g-1, respectively.
61
62
Prediction of further extraction results
63
Instead of performing lots of additional experiment, the obtained data are already sufficient
64
for further prediction of selectivity for MIP or NIP at different loaded amounts of flavonoids.
65
Based on the plot in Figure 2, the competitive adsorption isotherm-like relationship can be
66
established as
67
(1)
68
(2)
69
(3)
70
where
(y-axis in Figure 2) is mass of analyte i bound onto the MIP or NIP, and
71
Figure 2) is loaded mass of the analyte i. The subscript i is the index for flavonoids (QU, MO
72
and CA = quercetin, morin and catechin, respectively).
73
analyte i varied with the used polymer (MIP or NIP). These
74
the values resulting in the best fit to the experiment. With the unit of
75
i per g of polymer”, and the unit of
76
using least square fitting procedure, as shown in Table S4. By using these
77
and equation 1-3, flavonoid amount bound onto (and thus selectivity of) the MIP for any
78
flavonoids tested can be reliably predicted from any given loaded amounts of flavonoids. The
79
plots between calculated and experimental values are in Figure S1.
and
and
(x-axis in
are fitting constant for
values are nothing than
being “mg of analyte
being “mg”, the corresponding values were achieved
80
81
3
and
values
82
Table S1. Extraction performance and total binding capacity of three flavonoid standards
83
without extraction (direct analysis) and with extraction from different modified micropipette
84
tips prepared with two different monomers (MA and 4VP), number of experiments n = 3.
85
Note that the reused MA-MIP tip was cleaned with 10% v/v acetic acid in ethanol (100 µL)
86
followed by absolute ethanol (100 µL) and equilibrated with water (50 µL) prior to each
87
repeated extraction experiment.
88
Catechin
Morin
Quercetin
50.00
50.00
50.00
Flow through fraction (× 10-4 mg)
9.1 ± 0.1
4.9 ± 0.3
2.5 ± 0.2
Eluted fraction (× 10-4 mg)
21.6 ± 0.2
27.6 ± 0.5
31.5 ± 0.8
Binding capacity (mg g-1 polymer) a
0.82
0.90
0.95
% Bound b
43.2
55.2
63.0
Flow through fraction (× 10-4 mg)
17.8 ± 0.7
15.4 ± 0.8
23.0 ± 0.8
Eluted fraction (× 10-4 mg)
3.3 ± 0.3
0.0
0.0
Binding capacity (mg g-1 polymer) a
0.64
0.69
0.54
% Bound b
6.6
0.0
0.0
Flow through fraction (× 10-4 mg)
28.8 ± 1.4
0.02 ± 0.0
31.9 ± 1.4
Eluted fraction (× 10-4 mg)
6.8 ± 0.7
20.7 ± 0.8
14.2 ± 1.1
Binding capacity (mg g-1 polymer) a
0.42
1.00
0.36
% Bound b
13.7
41.4
28.5
Flow through fraction (× 10-4 mg)
31.3 ± 0.8
5.2 ± 0.3
35.1 ±0.5
Eluted fraction (× 10-4 mg)
0.0 ± 0.1
7.6 ± 0.2
0.0
Binding capacity (mg g-1 polymer) a
0.37
0.90
0.30
% Bound b
0.0
15.2
0.0
Direct Analysis (× 10-4 mg)
MA-MIP
MA-NIP
4VP-MIP
4VP-NIP
89
90
91
a
b
Binding capacity = (Direct Analysis – Flow through fraction) / Amount of MIP or NIP used
% Bound = (Eluted fraction)  100 / Direct analysis
4
92
Table S2. Extraction performance, total binding capacity and recovery percentages of three
93
flavonoid standards without extraction (direct analysis) and with extraction from different
94
modified micropipette tips prepared with MA monomer.
95
Catechin
Morin
Quercetin
37.5
37.5
37.5
Flow through fraction (× 10-2 mg)
33.7
22.5
19.0
Eluted fraction (× 10-2 mg)
3.7
14.5
16.8
Binding capacity (mg g-1 polymer) a
7.7
30.0
37.0
% Recovery b
99.8
98.8
95.4
Flow through fraction (× 10-2 mg)
33.8
22.5
25.4
Eluted fraction (× 10-2 mg)
3.5
14.3
10.9
Binding capacity (mg g-1 polymer) a
7.5
24.3
21.0
% Recovery b
99.4
98.1
96.8
Direct Analysis (× 10-2 mg)
MA-MIP
MA-NIP
96
97
98
a
b
Binding capacity = (Direct Analysis – Flow through fraction) / Amount of MIP or NIP used
% Recovery = (Flow through fraction + Eluted fraction)  100 / Direct analysis
99
100
101
Table S3. Comparison of binding capacity a in different platforms.
MIP Extraction Platform b
102
103
104
105
106
107
108
109
110
Catechin
Morin
Quercetin
Monolith-in-tip (mg g-1 polymer)
0.82
0.90
0.95
Packed tip (mg g-1 polymer) c
0.64
0.81
0.84
Batch extraction (mg g-1 polymer) c
0.24
0.29
0.39
Binding capacity for monolith-in-tip or packed tip format = (Direct Analysis – Flow through fraction) /
Amount of MIP used. Binding capacity for batch extraction = (Direct Analysis – Supernatant fraction) /
Amount of MIP used.
b
All experiments were loaded with 50 mg L-1 (100 µL) standard solution (direct analysis) and 5 mg of MA-MIP.
Batch extraction was conducted by suspending MA-MIP (5 mg) in the standard solution (50 µL) for 2 h, before
supernatant fraction was further analysed.
c
The prepared monolith MA-MIP was ground to fine particles before the performance test of packed tips and
batch extraction.
a
111
112
113
5
114
Table S4. Fitting constants according to the equation S1-S3 as well as the corresponding least
115
square values for the prediction of flavonoid extraction performance of each polymer
116
modified tip.
117
Catechin
Fitting constant
Morin
Quercetin
MA-MIP
MA-NIP
MA-MIP
MA-NIP
MA-MIP
MA-NIP
Ai
92.2
106.8
286.7
262.2
340.7
251.0
Bi
2.9
6.2
1.6
1.5
1.8
1.4
Least square value
0.64
0.27
0.49
0.95
0.92
0.95
118
119
120
121
A
B
30
20
10
0
0.0
0.1
0.2
0.3
0.4
Loaded amount (mg)
C
40
Amount bound (mg g-1)
Amount bound (mg g-1)
40
30
20
10
0
0.0
0.1
0.2
0.3
0.4
Loaded amount (mg)
Amount bound (mg g-1)
40
30
20
10
0
0.0
0.1
0.2
0.3
0.4
Loaded amount (mg)
122
123
Figure S1. Experimental extraction yield curves of MIP- (●) and NIP- () modified tips with
124
the predicted trends of the MIP (blue line) and NIP (red line), by using equations S1-S3, for
125
(A) catechin, (B) morin and (C) quercetin.
126
6
127
A
x10
eCA
4
2.0
x10
1.5
0.8
0.5
0.4
0.0
0.0
17.8
Abundance
1st
1.2
CA
1.0
x10
QU
2
1.6
18.6
19.4
20.2
21.0
21.8
21.5
23.5
Time (min)
5
25.5
27.5
Time (min)
0.8
0.6
0.4
0.2
0.0
10
12
14
16
18
20
22
24
26
Time (min)
B
x10
eCA
4
2.0
x10
1.5
0.8
0.5
0.4
0.0
0.0
17.8
Abundance
2nd
1.6
1.2
CA
1.0
x10
QU
2
18.6
19.4
5
20.2
21.0
21.8
21.5
23.5
Time (min)
25.5
27.5
Time (min)
0.8
0.6
0.4
0.2
0.0
10
12
14
16
18
20
22
24
26
Time (min)
C
x10
eCA
4
2.0
x10
1.5
1.2
1.0
0.8
CA
0.5
0.4
0.0
0.0
Abundance
17.8
x10
3rd
QU
2
1.6
18.6
19.4
20.2
21.0
21.8
21.5
23.5
Time (min)
5
25.5
27.5
Time (min)
0.8
0.6
0.4
0.2
0.0
10
12
14
16
18
20
22
24
26
Time (min)
128
129
Figure S2. GCMS results of three repeated eluted fraction 3 from the quercetin imprinted
130
MA-MIP by loading with the same tea sample solution; 1st, 2nd and 3rd replicates being A-C,
131
respectively.
132
133
7