Supporting Information
1 Synthesis of PAMAM-grafted silica nanoparticle
Figure S1. The synthetic routes for PAMAM grafted-silica nanoparticles
The PAMAM grafted-silica nanoparticles were obtained through the following steps:
(1) introduction of the amino groups onto the silica nanoparticle surface using
3-aminopropyltriethoxysilane (APES) as coupling agent; (2) Michael addition of
methylacrylate (MA) to amino groups on the SNP surface; (3) amidation of the
terminal ester groups with ethylenediamine (EDA).
1.1 Preparation of SNP-PAMAM G 0
The mixture of 5.0 g of silica nanoparticle, 300 mL toluene and 50 mL APES was
1
refluxed for 8 h under stirring, then was centrifuged for 20 min at 4000 rpm. The
precipitate was dispersed into 300 mL ethanol, sonicated for 30 min. After the
centrifugation-dispersion-sonication process was repeated five times, the product was
dried under vacuum at 50 °C for over 48 h.
1.2 Preparation of SNP-PAMAM G 0.5 (Michael addition)
The mixture of 3.0 g SNP-PAMAM G 0, 100 mL methanol and 0.5 mL MA was
reacted at 50 °C for 48 h. After reaction, the mixture was washed five times with
methanol following the method described in Section 1.1.
1.3 Preparation of SNP-PAMAM G 1.0 (amidation of terminal eater groups)
The mixture of 3.0 g SNP-PAMAM G 0.5, 100 mL methanol and 2.2 mL EDA was
reacted at 50 °C for 48 h. After reaction, the mixture was washed five times with
methanol.
Higher generations were obtained by repeating steps 1.2 and 1.3.
2
2 FTIR analysis
Fourier transform infrared (FTIR) spectra in the range of 4000 cm-1 – 400 cm-1 were
obtained after 20 scans, at a resolution of 2 cm-1, on an IRAffinity-1
spectrophotometer (Shimadzu, Kyoto, Japan). Prior to analysis, 1.5 mg NP sample
and 150 mg KBr were dried under an infrared lamp for 15 min. Afterward, they were
mixed thoroughly and crushed using a mortar and pestle, and then were placed in a
pellet die to form a KBr disc via applying pressure.
Compared with SNPs (trace 1 of Figure S2), new absorbing bands at 2926 cm-1 and
2850 cm-1, generated by the asymmetric and symmetric stretching vibrations of -CH2,
respectively, were observed with all SNP-PAMAMs. In the spectra from the
full-generation products, new absorption at 1640 cm-1 and 1554 cm-1 appeared, which
were respectively attributed to C=O stretching and N-H bending of amide groups
(-CONH-). The absorption bands centered at 1735 cm-1, clearly visible in the spectra
of all the half-generation products, generated by the C=O stretching of the ester
groups, disappeared after reacting with EDA to form the corresponding
full-generation SNP-PAMAMs. The IR results suggested that different generations of
PAMAMs were successfully grafted on the SNP surfaces.
3
Figure S2. FTIR spectra of SNPs grafted with different generation PAMAMs.
1. SNPs; 2-8, SNP-PAMAMs of different generations: 2, G 0; 3, G 0.5; 4, G 1.0; 5, G
1.5; 6, G 2.0; 7, G 2.5; 8, G 3.0.
3 TGA analysis
Figure S3. TGA curves of SNPs grafted with PAMAMs of different generations: A, G
0; B, G 0.5; C, G 1.0; D, G 1.5; E, G 2.0; F, G 2.5; G, G 3.0.
TGA curves of all the samples showed a slight weight loss below 120 °C, arising from
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the loss of adsorbed water from the samples [1]. Loss of the grafted organic groups
took place mainly from 120 °C to 600 °C, within which two weight loss stages could
be identified. The weight loss between 120 °C and 350 °C corresponded to the
decomposition of PAMAM moieties, while the one between 350 °C and 600 °C was
predominantly caused by the degradation of the APES-grafted SNP core [2]. The
increasing weight loss with higher generation SNP-PAMAMs suggests the growth of
hyperbranched PAMAMs on the SNP surface.
5
Table S1. Weight losses of organic groups calculated from TGA traces for different
samples
%Wt. change
Observed
%Wt. loss
Theoretical
compared to
Samples
Observed
Theoretical
amino
in range
amino groups
the proceeding
PG %
PG %
content
120-600°C
(mmol/g)
sample
(mmol/g)
SNP-PAMAM G 0
7.60
8.2
0.66
SNP-PAMAM G
18.40
10.80
22.6
23.2
19.28
0.88
23.9
29.1
24.12
4.84
31.8
58.7
26.02
1.90
35.2
76.3
28.50
2.48
39.5
89.8
31.36
2.86
45.7
121.6
0.5
SNP-PAMAM G
0.34
1.31
0.74
2.62
1.62
5.24
1.0
SNP-PAMAM G
1.5
SNP-PAMAM G
2.0
SNP-PAMAM G
2.5
SNP-PAMAM G
3.0
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3.1 Calculation of observed amino groups on the surface of full generation PAMAM
grafted-SNPs
3.1.1 SNP-PAMAM G 0
The weight loss of SNP-PAMAM G 0 in the range 120-600 °C was 7.60 g organics/
100 g modified nanoparticles (76 mg/1 g nanoparticles). Taking molecular weight of
116 g/mol and one primary amino group per molecule for G 0, the content of amino
group of SNP-PAMAM G 0 is 0.655 mmol/g.
3.1.2 SNP-PAMAM G 1.0
The amino group underwent Michael addition from G 0 was the differential wet loses
between SNP-PAMAM G 0.5 and SNP-PAMAM G 0 in the rang of 120-600 °C,
which was 10.8%. Because the molecular weight difference between G 0 and G 0.5 is
172, the content of amino groups reacted was calculated to be 0.628 mmol/g.
Therefore, the amount of unreacted amino groups was 0.655-0.628 = 0.027 mmol/g.
Similarly, based on the weight lose difference of 0.88% between SNP-PAMAM G 1.0
and SNP-PAMAM G 0.5, and the molecular increase of 56 during the amidation
reaction, one can deduce that 0.314 mmol/g amino group was generated from
SNP-PAMAM G 0.5 to SNP-PAMAM G 1.0. Therefore, the ultimate amino group
content on the surface of SNP-PAMAM G 1.0 was 0.314 + 0.027 = 0.341 mmol/g.
By using similar calculations, the amino group on the SNP-PAMAM G 2.0 and
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SNP-PAMAM G 3.0 were 0.738 mmol/g and 1.620 mmol/g, respectively.
3.2 Calculation of theoretical amino groups on the surface of full generation PAMAM
grafted-SNPs
Assuming each reaction after SNP-PAMAM G 0, amino group content of which was
0.665 mmol/g, was finished stoichiometrically completely, The amino groups of
SNP-PAMAM G 1.0 should be 0.655 mmol/g×2 = 1.310 mmol/g; the amino groups
of SNP-PAMAM G 2.0 should be 0.655 mmol/g×4 = 2.620 mmol/g; and the amino
groups of SNP-PAMAM G 3.0 should be 0.655 mmol/g×8 = 5.240 mmol/g.
3.3 Calculation of the grafting percentage (%) of the modified nanoparticles
Using the formula grafting percentage (%) = weight loss percentage /(100%-weight
loss percentage), the grafting percentages of SNP-PAMAMs were calculated to be
8.22%, 22.55%, 23.89%, 31.78%, 35.20%, 39.50% and 45.69%, respectively, for G 0,
G 0.5, G1.0, G 1.5, G 2.0, G 2.5 and G 3.0.
3.4 Calculation of theoretical grafting percentage (%) of different PAMAM-modified
nanoparticles
The calculations were based on the assumptions that, after the alkylation of silica
surface (G 0, amino group content 0.655 mmol/g) each subsequent reaction took place
stoichiometrically completely. For example, production of SNP-PAMAM G 0.5
resulted in an increase of 172 Dalton to every amino group; therefore, the mass of
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organic material grafted was 0.655 × (172+116) /1000 = 0.1886 g, and the theoretical
grafting percentage is 0.1886 /(1-0.1886) = 23.24%.
Similarly, the theoretical grafting percentage for SNP-PAMAMs at G 1.0, 1.5, 2.0, 2.5
and 3.0 are 29.08%, 58.70%, 76.27%, 89.82% and 121.58%, respectively.
3.5 Calculation of silanol groups on the surface of SNPs
It was widely assumed that the density of silica spheres is 2.0 g/cm3. The volume of
20-nm silica sphere is
4/3 πr3 = 4.19×10-24 m3
The mass of each silica sphere = 2.0 /10-6 g/ m3×4.19×10-24 m3 = 8.38×10-18 g
One gram silica spheres contains 1 g /8.38×10-18 g = 1.19×1017 silica spheres
The area of one sphere: 4 π r2 = 4 π (20/2nm) 2 = 1256 nm2
The density of silanol groups: 4.6/nm2
The number of silanol groups on one silica sphere: 4.6×1256 = 5.78×103
Total silanol groups on the surface of 1g SNP: 5.78×103×1.19×1017 spheres =
6.88×1020 = 1.14 mmol
The unreacted silanol groups on the surface of 1 g sample: 1.14 mmol/g - 0.655
mmol/g = 0.485 mmol/g
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3.6 Calculation of the net charges on the SNP-PAMAMs surface
We take pH 9.1 (the pH value of the optimum buffer described in Section 3.3 of the
main text, i.e., 12.5 mM borate/phosphate) as example to calculate the net charges of
SNP-PAMAMs. There are two kinds of amino groups in a PAMAM structure, primary
and tertiary, with protonation constants (pKs) of ca. 9.0 and 7.0, respectively [3].
Because the tertiary amino groups nearly fully deprotonate at pH 9.1, we consider the
primary amino groups the only potential proton acceptors for simplicity. In Section
3.1, the content of grafted primary amino groups increased from 0.34 mmol/g with G
1.0 to 1.62 mmol/g with G 3.0. On the other hand, the reported population data of
silanol groups on the silica surface and the protonation/deprotonation equilibrium
constants vary widely [4-6], so we can only semiquantitatively assess the residual
silanol content and, therefore, the net charge on the SNP-PAMAM surface. By
assuming the silica nanoparticle density to be 2.0 g/cm3 [7] and silanol population
density on the silica surface 4.6/nm2 [8], the content of residual silanol groups on the
grafted SNPs was estimated to be 0.485 mmol/g from the TGA results. According to
references [5, 6], there exist at the water/silica interface two kinds of silanol groups,
isolated and vicinal, possessing pKa values of 4.9 and 8.5, with corresponding surface
populations of 19% and 81%, respectively. So, the content of protonated silanol
groups was calculated to be 0.406 mmol/g. Similarly, a protonated silanol content of
0.303 mmol/g could be obtained based on the data of pKas as well as the isolated and
the vicinal silanol populations provided in another reference [9], in which the above
values were 5.5 and 9, 15% and 85%, respectively.
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Based on the parameters in Ref [6], the net charges SNP-PAMAMs at G 0, 1.0, 2.0
and 3.0 were calculated to be:-0.12 mmol/g, -0.26 mmol/g, -0.08 mmol/g, +0.31
mmol/g. Likewise, based on Ref [9], the net charges were -0.01 mmol/g, -0.15
mmol/g, +0.02 mmol/g, +0.41 mmol/g.
4 The equilibrium of the nanoparticles to the internal capillary wall
Figure S4. The EOF change during equilibration of capillary.
Between runs, the capillary was rinsed with running buffer. The EOF mobilities with
migration times were determined directly from the migration time of DMSO (the EOF
marker). Conditions: buffer, 10 mM sodium tetraborate and 2.5 mM sodium
dihydrogen phosphate, pH=9.1 containing 0.01% SNP-PAMAM G 2.0.
As judged from the variation tendency of EOF in Fig. S4, an equilibration time of
about 50 min was needed.
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5 Influences of PAMAM G 1.0 on the separation of proteins at different pHs
Figure S5. Influences of PAMAM G 1.0 on the separation of proteins at different pHs
(A) pH=10; (B) pH=9.3; (C) pH=9.15; (D) pH=8.8; (E) pH=8. Peak identification: 1,
Lyz; 2, Cyto C
Fig. S5 indicates that the second peak, Cyto C, could be observed when the buffer pH
was elevated to 9.3 or 10; however, GG and Myo were not detected in 60 min. The
phenomena imply the differences between grafted- and conventional PAMAMs.
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6 Variation of theoretical plate numbers as a function of SNP-PAMAM G 2.0
concentration
Table S2. Influence of SNP-PAMAM G 2.0 concentration on theoretical plate
numbers
Concentration
Plate number
(%)
Lyz
Cyto C
GG
Myo
0
N/A
N/A
25622
2405
0.002
N/A
N/A
131631
5861
0.004
N/A
N/A
149424
7859
0.006
2624
3836
145349
12119
0.008
3856
9411
204806
4792
0.010
5557
13616
170003
23116
0.012
4396
10446
66605
N/A
The theoretical plate number (N) of a peak was calculated using the formula
N  5.54(
t 2
)
w0.5
where t is the migration time, W0.5 is the peak width at the half height.
The numbers of theoretical plates, within experimental errors, noticeably increased
with SNP-PAMAM concentration until 0.010%, at which the highest theoretical plate
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numbers were obtained for three proteins.
14
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