Smart Porous Monoliths for Chromatographic Separations
3.2 Temperature Responsive Polymers
3.2.2 Silica and Hybrid Monoliths
copolymer. Separation of the three analytes was obtained in the column polymerized for 4 h (Figure 3.6c). Although separation was achieved, the chromatographic efficiency was very poor (very wide peak widths) in comparison with the separations found in the PNIPAAm‐co‐MBAAm column, or with P(MEO2MA‐co‐OEGMA) grafted on a silica support [36], as will be discussed later. This performance may be assigned to the low surface areas and absence of mesopores in the totally organic polymer.
Smart Porous Monoliths for Chromatographic Separations 83
better efficiency in terms of number of plate numbers and peak symmetry, but it was not able to separate hydrocortisone and prednisolone because of the very close hydro- phobicity of these steroids. This separation in the smart monolith was attributed to interactions with some polar groups (N─H and C═O), which could be exposed to the mobile phase, even in the shrunk conformation of PNIPAAm at 55 °C, thus interacting with the polar groups of the steroids.
0.6
(a) (b)
(c) (d)
1
1
1 2
2
3
2 + 3 2 + 3
1 12 10 8 6 4 2
0 0 5 10 15 20 25 30
0.3 0.0 –0.3 –0.6 –0.9 –1.2 0
24 18 12 6 0
–60 3 6 9 12
2 4 6
Retention time (min) Retention time (min)
Absorbance (mV) Absorbance (mV)
Retention time (min)
35 28 21 14 7
00 5 10 15 20 25
Absorbance (mV)
Retention time (min)
Absorbance (mV)
8 10 12
Figure 3.6 Elution profiles of aqueous mixtures of the steroids, (1) hydrocortisone, (2) testosterone, and (3) medroxyprogesterone acetate, at 40 °C (1 ml min−1) on (a) ungrafted column, (b–d) grafted for 3, 4, and 8 h, respectively, using a [MeO2MA]/[OEGMA] ratio of 85/15. Source: Reprinted from Reference [35]. Copyright 2013 American Chemical Society. Reproduced with permission of the American Chemical Society.
+ CH2=CH
C=O
NH HO HO
O
CN HN O
S
n
S NC
S S
RAFT agent
AIBN;DMF 70 °C
PNIPAAM-COOH O
NIPAAM
Figure 3.7 RAFT polymerization of NIPAAM using CPDB as a chain transfer agent. Source: Reproduced from Reference [38]. Reproduced with permission of Elsevier.
Roohi et al. [39] investigated the influence of the molar mass of the polymers on the separation performance of grafted silica beads in the separation of steroids. Control of the polymerization time of a mixture of NIPAAM, DDAT (RAFT agent), and AIBN in DMF allowed production of PNIPAAm with molar masses of 4200, 8500, and 13 800 g mol−1. These polymers were activated with N‐hydroxysuccinimide and then were attached to the aminated silica surfaces. The hydrophobicity of the columns grafted with the 4200 g mol−1 PNIPAAm was not high enough to distinguish between hydrocortisone and prednisolone. The other two grafted columns separated all five steroids, but the peaks obtained in the column grafted with the 13 800 g mol−1 PNIPAAm exhibited significant tailing because of diffusion of the analytes into the thicker layer of
200 nm 200 nm
50 nm 50 nm
(a) (b)
(c) (d)
Figure 3.8 SEM and TEM images of pure silica (a, c) and the final thermoresponsive composite (b, d).
Arrows in (c) and (d) show the free mesopores. Source: Reproduced from Reference [38]. Reproduced with permission of Elsevier.
35 1,2,3,4
5 5 5
1,2,3,4
5 °C 15 °C
1,2,3,4
25 °C 30
25 20 15 10 5 0
2 4 6
Retention time (min)
Intensity
8 10 12
35 80
70 60 50 40 30 20 10 0
2 3 4 5 6
30 25 20 15 10 5 0
2 4 6
Retention time (min) Retention time (min)
Intensity Intensity
8 10 12
30 14 6
5 4 3 2 1 0
2 4 6 8 10 12
12 10 8 6 4 2 0
2 4 6 8 10 12
2,3,4
35 °C 45 °C 55 °C
1 43
14 3
2 5
2 5
1 25 20 15 10 5 0
2 3 4 5
7.0 Hydrocortisone
Hydrocortisone acetate Dexamethasone Prednisolone Testosterone
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
10 20 30
Temperature (°C)
40 50 60
Retention time (min) Retention time (min)
Retention time (min)
Retention time (min)
Intensity Intensity Intensity
Figure 3.9 Chromatograms and change of retention time with temperature of an aqueous mixture of steroids at different temperatures in a PNIPAAm grafted (100 × 4.6 mm i.d.) silica monolithic column. (1) Hydrocortisone, (2) hydrocortisone acetate, (3) dexamethasone, (4) prednisolone, and (5) testosterone. Mobile phase: water; sample volume = 10 μl; flow rate = 1 ml min−1. Source:
Reproduced from Reference [38]. Reproduced with permission of Elsevier.
polymer. The silica materials grafted with 8500 g mol−1 PNIPAAm were used to com- pare the separation performance in packed beads and in monolithic format (all columns with 100 × 4.6 mm dimensions). The monolithic column exhibited the best separation performance, a fact explained by the high packing density of the silica beads resulting in less free volume for interaction between the analytes and the polymer chains.
Additionally, the presence of only macro pores in the silica beads was not enough for efficient separations, with the best performance being achieved by the columns affording a hierarchical distribution of meso and macro pores [39].
Nagase et al. [40] used a different approach to graft PNIPAAm brushes to the surface of monolithic silica. Instead of preparing the PNIPAAm in solution and then attaching the ATRP agent to the polymer, these authors proposed a surface‐initiated ATRP approach by first grafting an ATRP initiator, ((chloromethyl)phenylethyl)trimethoxysi- lane (CPTMS), to the silanol groups of the silica monolith. To grow the PNIPAAm brushes on the RAFT‐agent modified silica monolith, a solution of NIPAAm was pre- pared in 2‐propanol. After the solution was deoxygenated, CuCl, CuCl2, and tris[2‐
(N,N‐dimethylamino)ethyl]amine (Me6TREN) were added under a nitrogen atmosphere, and the solution was then pumped through the silica monolith for 16 h (0.050 ml min−1) (Figure 3.10). This strategy increased the amounts of grafted PNIPAAm on the silica surfaces compared with those prepared by the conventional radical polymerization.
The effect of the density of the PNIPAAm on the separation of steroids was investigated (Figure 3.11). The density of PNIPAAm brushes was decreased by grafting the monolith with a mixture the RAFT agent and (3‐glycidoxypropyl)trimethoxysilane, which binds to the silanol groups but does not bind to the PNIPAAm brushes (Figure 3.10b).
Modifications were also made in 5 μm silica beads, which were then packed in a stain- less‐steel column. The monolithic columns exhibiting both high and low density of PNIPAAm separated the mixture of steroids at a temperature above LCST using water as mobile phase. Additionally, the monolithic structures enabled faster and more effi- cient separation than the particle packed columns. Whereas the high density PNIPAAm columns exhibited higher retention, the low density columns allowed faster separations [40] (Figure 3.11).
The strategy of grafting the RAFT initiator to the surface of the silica monolith was explored further to produce thermally modulated cationic smart monoliths [41]. Thus, CPTMS grafted silica was used as support to grow thermoresponsive polymeric brushes from a mixture of NIPAAm, N,N‐dimethylaminoethyl methacrylate (DMAEMA) and N‐tert‐butylacrylamide (tBAAm) monomers mixed with the catalytic ATRP system composed of CuCl, CuCl2, and Me6TREN. The LCST of the resulting P(NIPAAm‐co‐
DMAEMA‐co‐tBAAm) in phosphate buffer (pH 7.0) was 41.4 °C with a pKa of 7.94 (at 4 °C) [41]. This column was able to separate adenosine monophosphate (AMP), adeno- sine diphosphate (ADP), and adenosine triphosphate (ATP) by isocratic elution in phosphate buffer at 10, 30, and 50 °C. Retention times decreased with the increasing temperature, as expected, because of the higher hydrophobicity at temperatures >LCST.
Additionally, at 50 °C the separation between AMP and ADP was not achieved at base line, as observed for the temperatures <LCST, where the ion exchange mechanism dominated the separation. Comparison of the monolithic and packed silica beads grafted to P(NIPAAm‐co‐DMAEMA‐co‐tBAAm) showed the best performance of the monolithic structure for separation of acidic bioactive compounds [41]. A similar strat- egy for producing anionic copolymers was already presented for silica beads using the 2‐acrylamido‐2‐methylpropanesulfonic acid monomer [42].
3-D silica-rod ATRP-initiator
ATRP-initiator
Monolithic silica-rod surface ATRP-initiator modified surface Dense PIPAAm brush grafted surface
Monolithic silica-rod surface ATRP-initiator modified surface Dilute PIPAAm brush surface GPTMS
CH3O CH3O (CH2)2
CH2Cl Si
CH3O CH3O
CH3O (CH2)3 O CH2 CH O
CH2 CH3O Si
CH3O CH3O (CH2)2
CH2Cl Si
CH3O CH3O CH3O (CH2)2
CH2Cl Si
CH3O CH3O CH3O (CH2)2
CH2Cl Si
CH3O CH3O CH3O (CH2)2
CH2Cl
Si O (CH2)2
CH2Cl Si
O (CH2)2 CH2Cl Si
O O O
(CH2)2 CH2Cl Si
O (CH2)2
CH2 Cl
= IPAAm monomer
Cl
Cl Si
O (CH2)2 CH2 Si
O O O
(CH2)2 CH2 Si
O (CH2)2 CH2Cl Si
O O
O (CH2)3 CH2CHCH2 Si
O O O
(CH2)2 CH2Cl Si
O Si(CH2)2 CH2
O O
O (CH2)3 CH2 CH2
Cl
Cl CH
Si
O O O
(CH2)2 CH2 Si
CH3O OH
(a)
(b)
in toluene 25 °C, 16 h
in 2-propanol 25 °C, 16 h IPAAm CuCl CuCl2 Me6TREN Flowing at 0.05 ml min–1
in 2-propanol 25 °C, 16 h IPAAm CuCl CuCl2 Me6TREN Flowing at 0.05 ml min–1 Circulating
at 0.1 ml min–1
in toluene 25 °C, 16 h Circulating at 0.1 ml min–1 OH
OH
OH
OH
OH
Figure 3.10 Preparation of PNIPAAm brush grafted monolithic silica‐rod surfaces by ATRP; (a) dense PNIPAAm brush surface and (b) dilute PNIPAAm brush surface. Source: Reprinted from Reference [40]. Copyright 2011 American Chemical Society. Reproduced with permission of the American Chemical Society.
In another work, Nagase et al. [43] used CPTMS modified silica monoliths and beads to graft a copolymer of NIPAAm and butyl methacrylate (BMA) by ATPR. Incorporation of hydrophobic monomers such as BMA separate hydrophilic analytes via hydrophobic interactions. The molar ratios of NIPAAm to BMA varied from 90 : 10 to 100 : 0 and the reaction times were 4 or 16 h, with longer polymerization times producing longer copolymer brushes. At a molar ratio of 93 : 7 the LCST decreased from 32.1 to 8.9 °C in the P(NIPAAm‐co‐BMA) column. The columns were tested for separation of sodium benzoate, phenol, methylbenzene, ethyl p‐aminobenzoate, ethyl benzoate, and methyl hydroxybenzoate. Base line separations of the six compounds were observed at 50 °C, proving that the hydrophobic interaction dominates the separation mechanism. At 30
°C, improved efficiency was observed in the columns affording shorter copolymer chains (polymerized for 4 h), a fact explained by the longer time needed for diffusion of the analytes through the thicker polymer layer obtained with 16 h of polymerization.
Again, the monolithic format performed better than packed silica particles treated by similar protocols to graft PNIPAAm‐co‐BMA. Insulin chain A, insulin chain B, and insulin were also successfully separated in the monolithic columns [43].
Despite the extensive use of PNIPAAm, this polymer is not biologically inert, contain- ing secondary amide functions available for hydrogen bonding with peptides and pro- teins, thus affecting their chromatographic separations. To overcome this potential limitation of PNIPAAm, oligo(ethylene glycol)‐based thermoresponsive polymers have
1
1234
1 1
1
1
1 1 1
0 (a)
(b)
(c)
2 4
Time (min)
6 0 2 4
Time (min) 6
8 10 0 10 20
Time (min)
30 40
2
2
2
2
2 23
23 4 5
2 3
3
3
3
3 4
4
3 4 4
4
4 4
5
5
5 30 °C
10 °C
10 °C
10 °C
30 °C
50 °C 30 °C
50 °C 50 °C
5 5
Figure 3.11 Chromatograms of a mixture of (1) hydrocortisone, (2) prednisolone, (3) dexamethasone, (4) hydrocortisone acetate, and (5) testosterone on (a) dense PNIPAAm‐brush grafted monolithic silica‐rod column (50 × 3.2 mm i.d.), (b) dilute PNIPAAm‐brush grafted silica‐rod column, (50 × 3.2 mm i.d.), and (c) dense PNIPAAm‐brush grafted silica beads column (50 × 4.6 mm i.d.). Mobile
phase = water; flow rate = 1 ml min−1. Source: Reprinted from Reference [40]. Copyright 2011 American Chemical Society. Reproduced with permission of the American Chemical Society.
Smart Porous Monoliths for Chromatographic Separations 89
been proposed [35, 36] because they are mainly composed of bioinert ethylene oxide units. Copolymers of MeO2MA and OEGMA were prepared from the commercially available monomers by ATRP in the presence of the initiator N‐succinimidyl‐2‐
bromoisobutyrate [36]. These P(MEO2MA‐co‐OEGMA) copolymers were attached to aminated monolithic silica via standard amide coupling chemistry through the N‐succinimidyl ester chain ends. The LCST of these polymers depended on the [OEGMA]/[MEO2MA] ratio. The potential application as a temperature driven chro- matographic column was proven by separation of five steroids at diverse temperatures.
As in the work of Li et al. [35], who grafted P(MEO2MA‐co‐OEGMA) on a methacrylate monolith, the dependence of the separation on the temperature was not as evident as in the PNIPAAm grafted columns (Figure 3.12). Even so, an increase in temperature increased the separation efficiency using pure water as mobile phase, thus indicating that the stationary phase became more hydrophobic. Additionally, the rigid silica sup- port for the grafting of P(MEO2MA‐co‐OEGMA) provided better separation efficien- cies than that obtained with the methacrylate support [35]. Separation of two proteins with close hydrophobicity was demonstrated with the most hydrophobic column ([OEGMA]/[MEO2MA] = 5 : 95); thus, whereas lysozyme and myoglobin co‐eluted at 5 °C, they were separated upon raising the temperature to 45 °C.
Some silica based materials affording temperature responsive properties and addi- tional molecular recognition abilities were described in the form of particle packed columns, but not yet in the monolith format. For instance, Liu et al. [44] prepared a column for capture and release of molecules containing cis‐diol functionalities exploit- ing the chemistry of boronic acid. For this, they treated 5‐μm 3‐aminopropyl silica par- ticles with 2‐bromoisobutyryl bromide (ATRP initiator) and then grafted the functionalized silica with NIPAAm and 4‐vinylphenylboronic acid (VPBA) and were able to separate and enrich adenosine from a mixture with deoxyadenosine, as well as capture and release of horseradish peroxidase [44].
1
(a) (b) (c)
2
2 Time (min)
Intensity (mAU)
0
3 3, 4 1,2
4
4 5
5
1 2
2
Time (min)
Intensity (mAU)
0
3 3 4
1 2 4
6 4
5
Composite a
Composite d 5
12
2 3 1
Time (min)
Intensity (mAU)
0
3 3, 4
1 2 4
6
4 5 7
5
Composite f
Composite e 5 5 °C
35 °C
Figure 3.12 Separations of (1) hydrocortisone, (2) prednisolone, (3) dexamethasone,
(4) hydrocortisone acetate, and (5) testosterone on (a) P(MEO2MA‐co‐OEGMA) columns obtained with [OEGMA]/[MEO2MA] 5 : 95; (b) [OEGMA]/[MEO2MA] 10 : 90 (composite a) and [OEGMA]/[MEO2MA]
10 : 90 (composite d), 55 °C; and (c) [OEGMA]/[MEO2MA] 15 : 85 (composite f) and [OEGMA]/[MEO2MA]
5 : 95 (composite e), 35 °C. Mobile phase = water, flow rate = 1 ml min−1, 100 × 4.6 mm i.d. columns.
Source: Reprinted from Reference [36]. Copyright 2009 American Chemical Society. Reproduced with permission of the American Chemical Society.
Table 3.2 summarizes typical monolithic materials currently used for separations, extraction, sensing, and construction of micro valves for flow‐based systems of analysis, showing the predominance of the thermal stimulus, especially that of PNIPAAm, on the development of smart monoliths.