Smart Porous Monoliths for Chromatographic Separations
3.2 Temperature Responsive Polymers
3.2.1 Grafted on Organic Monoliths
PNIPAAm is the most studied temperature responsive class of polymers, having a LCST of 32 °C. Among the thermoresponsive N‐alkylacrylamide polymers, PNIPAAm has the sharpest phase transition in response to temperature changes across the LCST [20].
Below the LCST the polymer chains are hydrated and exhibit an expanded conforma- tion in water. As the temperature rises above the LCST the expanded chains suffer a reversible collapse and dehydrate due to formation of intermolecular hydrogen bonding between the carbonyl and NH groups. The polymer loses about 90% of its volume during this phase transition from a swollen hydrated state to a shrunken dehydrated hydrophobic state. Hydrophobicity is caused by the isopropyl groups located in the collapsed chains [20].
The hydrophilic–hydrophobic thermal driven transition may be explored not only in a binary approach for capture and release of analytes in solid phase extraction but also
using programmed temperature gradients as demonstrated by Kanazawa [21] for the separation of steroids using pure water as mobile phase.
Grafting a polymer monolith with PNIPAAm was first described in 1997 by Peters et al. [22]. A generic poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) monolith, P(GMA‐co‐EDMA), was prepared by free radical thermal polymerization of a mixture of GMA (24% v v−1), EDMA (16% v v−1) and cyclohexanol (porogenic solvent, 60% v v−1) containing 1% (m m−1) of azobisisobutyronitrile (AIBN) with regard to the mass of monomers. After the polymerization (55 °C, 20 h) the epoxy groups reacted with allyl amine (60 °C, 8 h). These columns were then divided into two sets. In the first, the poly- mer was grafted with 10% NIPAAm (60 °C, 20 h). A second set of columns was treated by a similar process, but the grafting was made with 9.9% of NIPAAm and 0.1% of MBAAm, the latter being a crosslinker used to control the swelling. The modified tem- perature responsive monoliths were tested as thermal gates and thermal valves and in thermally controlled chromatographic separations.
The thermal gate effect was proven by pumping pure water (1 ml min−1) through a 10‐mm thick monolithic disk grafted with only PNIPAAm immersed in a water bath at 40 °C. At this temperature, the chains shrank and the pores of the monoliths opened, thus allowing the water to flow through the column under negligible backpressure.
Then, the column was cooled to room temperature for hydration and swelling of PNIPAAm, thus filling the pores, increasing the pressure to >20 MPa. When the column was re‐immersed in the water bath at 40 °C, an immediate decrease in the pressure was observed. To prove the thermal valve behavior, the 10‐mm thick disk column grafted with PNIPAAm crosslinked with MBAAm was used. Crosslinking with MBAAm prevented the PNIPAAm chains completely filling the pores below the LCST. Even so the backpressure at any flow rate studied was systematically higher at 25 °C than that at 40 °C. Thus, under a constant pressure, one would control the flow rate through the column temperature [22].
Hydrophobic interaction chromatography (HIC) was used to separate carbonic anhy- drase from soybean trypsin inhibitor by isocratic elution using 1.4 mol l−1 ammonium sulfate as mobile phase. The 10‐mm thick disk column grafted with PNIPAAm crosslinked with MBAAm was immersed in a water bath (40 °C) and equilibrated with mobile phase. After injection of the mixture of proteins, the hydrophilic anhydrase car- bonic was quickly eluted from the column, while the trypsin inhibitor was strongly retained. After 10 min, the column temperature was lowered to 25 °C and the resultant hydrophobic–hydrophilic transition allowed the mobile phase to elute the hydrophobic trypsin inhibitor [22].
After this seminal work, several other approaches were described to explore the porous structure of monoliths as supports for smart polymers aiming at separations and extractions [23, 24]. Additionally, thermal valves in microfluidic devices [25–27], actuators [28], and biosensing based on thermally reversible immobilization of glucose oxidase on PNIPAAm [29] have been described.
Mittal et al. [30] investigated further if the fast swelling and deswelling of the PNIPAAm chains around the LCST might be hindered in the restricted space of the pore surface of monoliths. For this, crosslinked polystyrene latex was prepared as both free particles and monoliths. PNIPAAm was grafted by atom transfer radical polymeri- zation (ATRP) in both materials. Swelling and deswelling kinetics around LCST in the monoliths were slower than those at free particles. The slower kinetics, however, did
Smart Porous Monoliths for Chromatographic Separations 77
not compromise the use of porous monolithic structures as supports for chromatographic separations driven totally by temperature changes.
Polyacrylamide polymers can be directly used as smart monoliths as demonstrated by Liu et al. [31] who prepared a cryogel by copolymerization of NIPAAm with MBAAm and PEG 20000 (porogenic solvent) by in situ free‐radical redox cryo‐polymerization (−12 °C) in a stainless steel tube (100 mm × 4.6 mm i.d.). In comparison with the hydro- gels prepared at room temperature, the monolithic cryogels exhibited a larger swelling ratio around the LCST and reached the deswell equilibrium much faster. These col- umns were used to separate a mixture of six steroids (Table 3.1, Figure 3.2), achieving 56 600 plates per meter (plate height of 17.7 μm) for betamethasone 21‐acetate at 55 °C using pure water as mobile phase at 1 ml min−1. In another work [32], the P(NIPAAm‐co‐
MBAAm) monolith was used to separate three aromatic ketones (acetophenone, log P = 1.6; propiophenone, log P = 2.2; and butyrophenone, log P = 2.5) at 55 °C using water as mobile phase at 1 ml min−1. The aromatic ketones were retained by hydropho- bic interaction, as expected, with the elution order following their coefficient partition in the n‐octanol/water system (log P).
The emergence of omics, especially proteomics and metabolomics, demanded the miniaturization of chromatographic systems and columns, as reviewed by Desmet and Eeltink [33]. To demonstrate the applicability of PNIPAAm in a capillary LC system, the polymer was grafted on a copolymer of styrene and divinylbenzene, P(St‐co‐DVB), prepared in the capillary format (10–20 cm × 100 μm i.d.) in the presence of polydimethylsiloxane (PDMS, porogen), followed by surface‐initiated ATRP of NIPAAm (Figure 3.3). The synthesis of the parent monolith in the presence of adequate amounts of PDMS formed macro pores with a mean diameter of 10.3 ± 2.5 μm. Characterization of the grafted and parent polymers by scanning electron microscopy analysis indicated that the pore structure of the parent monolith was intact after grafting the PNIPAAm brushes. The thermoresponsive behavior of these capillary columns was demonstrated by the separation of cortisone and dexamethasone at temperatures between 10 and 40 °C (Figure 3.4a); neither compound was irreversibly retained, and they were eluted by water at any temperature. Whereas both cortisone and dexamethasone eluted in a sin- gle chromatographic peak at 10 °C, the retention of dexamethasone, the most hydro- phobic steroid, increased systematically with increasing temperature, leading to almost base line separation at 40 °C. An additional experiment with cortisone and testosterone demonstrated that testosterone, the more hydrophobic compound (Table 3.1), can be irreversibly retained at 35 °C, being completely separated from the hydrophilic corti- sone. For this separation a step temperature from 35 to 15 °C expanded the PNIPAm chains, freeing testosterone to be detected in the UV detector [34] (Figure 3.4b).
In addition to PNIPAAm thermoresponsive polymers, recent research showed that copolymers of 2‐(2‐methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) exhibit LCST values that can be tuned in the range 26–90 °C by varying the co‐monomer composition [35]. Their LCST values were less affected than PNIPAAm by ionic strength, concentration of the copolymer, and chain length. Li et al. [35] prepared porous polymer monoliths grafted with poly[2‐(2‐meth- oxyethoxy)ethyl‐methacrylate‐co‐oligo(ethylene glycol) methacrylate], P(MEO2MA‐co‐
OEGMA) by a two‐step ATRP method. The organic polymer monolith was synthesized inside 50 × 4.6 mm stainless steel columns in the first ATRP step from a mixture of EDMA, 2‐bromopropionate (EBP), cuprous bromide, methanol, and hexane.
Steroid Structure Molar mass (g mol−1) Log P Hydrocortisone
O
O HO
H H H
OH OH
362.46 1.47
Prednisolone
O HO
HO O OH H
H H
360.44 1.62
Dexamethasone
O HO
OH OH
H H
O
F
392.46 1.83
Hydrocortisone acetate
O
O O
O
OH CH3
H3C H3C
H H H HO
404.50 2.45
Testosterone
O H H
H
OH 288.42 3.32
Medroxyprogesterone
O
O H3C
H3C CH3
CH3 OH H
H H
344.49 4.11
Progesterone
O
O
H H
H
314.47 3.87
Beclomethasone dipropionate
O HO
H H Cl
O O
O O
O
521.04 3.1
Smart Porous Monoliths for Chromatographic Separations 79
400 300 200 100
–100 0
0 2 4
3,4,5,6
25 °C 1,2
6 t (min)
Absorption (mV)
8 10 12
300 250 200 150 100 50
–50 0
0 2 4
3,4,5
6 35 °C 1,2
6 t (min)
Absorption (mV)
8 10 12
180 140 120 160
100 80 60 40
–20 20 0
0 2 4
5
45 °C
1 2
34
6 6
t (min)
Absorption (mV)
8 10 12 14 16
100 80 60 40 20
–20 0
0 10
55 °C
1 2
34 6 5
20 t (min)
Absorption (mV)
30 40
500 400 300 200 100 0
0 2 4
3,4,5,6
15 °C 1,2
6
Absorption (mV)
8 10
t (min)
(a) (b)
(c) (d)
(e)
Figure 3.2 Chromatograms of a mixture of steroids on the temperature‐responsive P(NIPAAm‐co‐
MBAAm) column using water as mobile phase (1 ml min−1): (1) hydrocortisone, (2) cortisone acetate, (3) prednisolone acetate, (4) fluocinolone acetonide, (5) betamethasone 21‐acetate, (6)
beclomethasone dipropionate. Temperature: (a) 15, (b) 25, (c) 35, (d) 45, and (e) 55 °C. UV detection at 254 nm; sample volume = 5 μl. Source: Reproduced from Reference [31]. Reproduced with permission of Elsevier.
The mixture was homogenized and purged with Ar before addition of 1,1,4,7,7‐penta- methyldiethylenetriamine (PMDETA). Polymerization was carried out for 12 h at room temperature. After washing the column with methanol and water, the second ATRP step was carried out with a mixture of CuBr2, PMDETA, MEO2MA, and OEGMA in water, followed by addition of hydrazine. The [MeO2MA]/[OEGMA] molar ratios were 85 : 15, 90 : 10, or 80 : 20. These mixtures were pumped through the column for different times (0, 3, 4, and 8 h) at 35 °C (Figure 3.5).
LCST values of 25.7, 36.8, and 44.1 °C were found for the free P(MEO2MA‐co‐
OEGMA) polymers synthesized with the [MeO2MA]/[OEGMA] ratios of 85 : 15, 90 : 10, and 80 : 20, respectively. Separation of hydrocortisone, testosterone, and medroxyprogesterone acetate was tested in water as mobile phase at different tempera- tures around the LCST. The retention times increased with increase in their hydropho- bicities, which were proportional to the amount of MEO2MA in the polymerization mixture (Figure 3.6, Table 3.1), and the separation was driven by the hydrophobic–
hydrophobic interactions.
Additionally, longer retention times were achieved in the longer P(MEO2MA‐co‐
OEGMA) chains, whose sizes were controlled via the polymerization times. The non‐
grafted monolith retained irreversibly medroxyprogesterone acetate because the hydrophobic interaction between the steroid and the stationary phase was too strong.
Testosterone and medroxyprogesterone acetate eluted in a single peak from the column polymerized for 3 h with 85 : 15 [MeO2MA]/[OEGMA] because the short chain length of P(MEO2MA‐co‐OEGMA) produced insufficient interaction sites to separate the steroids. Unresolved peaks were also observed using the column polymerized for 8 h (longest chain length) because the steroids diffused into the thick layers of the grafted
(a) Preparation of polystyrene monolithic capillary
(b) Modification of the PSt surface with PNIPAAm
HO H O H
O
MPTS
styrene m/p-DVB
PDMS AIBN O O
O O
O Si
mO x y
y O
O O
O in toluene, Si
45 °C, 18 h
ZnCl2
x x x
CH2Cl
NIPAAm Me6TREN
CuCl CuCl2 in 2-propanol
r.t., 24h
nCl O NH in CMME,
40 °C, 24 h
in mesitylene 60 °C, 24 h Inner surface of
capillary
Polystyrene (PSt)
PSt monolith ATRP initiator-
Immobilized PSt PNIPAAm grafted PSt (PNIPAAm-PSt) monolith
monolith
Figure 3.3 Preparation of (a) polystyrene monolith inside a fused silica capillary and (b) a PNIPAAm grafted polystyrene P(NIPAAm‐PSt) monolithic capillary. Source: Reproduced from Reference [34].
Reproduced with permission of Elsevier.
0 5
Retention time (min) Retention time (min) 10
1,2 (a)
(b)
10 °C
1
3 15 °C
1 1
3 35 °C 3
35 °C
15 °C 1
2
20 °C
1 2
30 °C
1 2
40 °C
15 20
0
Retention time (min)
10 20 30
0 5
Retention time (min)
10 15 20
0 5 10 15 20 0 5
Retention time (min)
10 15 20
0
Retention time (min)
10 20 30 0
Retention time (min)
10 20 30
Figure 3.4 Chromatograms of mixtures of (a) cortisone (1) and dexamethasone (2) at different temperatures and (b) cortisone (1) and testosterone (3) at 15 and 35 °C, and stepping the temperature from 35 to 15 °C on capillary (100 μm) PNIPAAm grafted polystyrene columns. Concentrations of steroids = 25 μg ml−1; mobile phase = pure water; sample volume 0.14 μl; flow rate = 2 μl min−1. Source:
Adapted from Reference [34]. Reproduced with permission of Elsevier.
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.