Porous polymer monoliths adapted for sample preparation

Abstract

A porous polymer monolith comprises a polymer body having macroporous through-pores that facilitate fluid flow through the body and an array of mesopores adapted to bind from the fluid flow molecules of a predetermined range of sizes, wherein the surface area of the monolith is predominantly provided by the mesopores. Also disclosed is a method of making a porous polymer monolith. The method includes forming a polymer body by phase separation out of a solution containing at least a monomer, a crosslinker and a primary porogen, whereby the body contains multiple macroporous through-pores, wherein the solution further contains a secondary porogen comprising oligomers inert with respect to the monomer and cross-linker but chemically compatible with the monomer so as to form mesostructures within the polymer body during said phase separation, and washing the mesostructures from the body to provide an array of mesopores such that the surface area of the monolith is predominantly provided by the mesopores.

Claims

1. A porous polymer monolith comprising: a polymer body having macroporous through-pores that facilitate fluid flow through the body and an array of mesopores comprising hydrophobic sites, said mesopores adapted to bind from the fluid flow molecules of a predetermined range of sizes, wherein the surface area of the monolith is predominantly provided by the mesopores; wherein the mesopores are formed by presence of a secondary porogen during phase separation of the polymer body from a solution, the secondary porogen comprising oligomers inert with respect to the monomer and cross-linker but chemically compatible with the monomer so as to form mesostructures within the polymer body during said phase separation, wherein the secondary porogen has a molecular weight of not more than 5000; wherein the porous polymer monolith has a bimodal pore size distribution; and wherein the porous polymer monolith is surface grafted to provide a hydrophilic external surface.

2. A porous polymer monolith according to claim 1 wherein the mesopores have a pore size in the range 40-120 Å.

3. A porous polymer monolith according to claim 1 wherein the macroporous through-pores have a pore size in the range of 2-3, micron.

4. A porous polymer monolith according to claim 1, surface grafted to prepare a mixed-mode ion exchange functionality.

5. A porous polymer monolith according to claim 1 wherein greater than 65% of the surface area of the monolith is provided by the mesopores.

6. A porous polymer monolith according to claim 1 wherein at least 85% of the surface area of the monolith is provided by the mesopores.

7. A porous polymer monolith according to claim 1 wherein at least 90% of the surface area of the monolith is provided by the mesopores.

8. A porous polymer body according to claim 1, wherein the polymer body comprises styrene monomers.

9. A porous polymer body according to claim 8, wherein the polymer body consists of styrene monomers.

10. A porous polymer body according to claim 1, wherein the mesopores have a pore size in the range of 20-120 Å.

Description

EXAMPLES

Example 1

(1) The different porous polymer monoliths (abbreviated in this example as “monoliths”) constituting embodiments of the first aspect of the invention were synthesised by the process of the second aspect of the invention, using as secondary porogens, polystyrenes with respective molecular weights of 906, 1300 and 1681 respectively. To determine the optimum binding capacity and recovery of different analytes on the monoliths synthesised with the various molecular weight polystyrenes, the monoliths were probed with analytes of increasing molecular weight as follows:

(2) TABLE-US-00001 Analytes Molecular weight (g/mol) 3-nitroaniline 138 peptide NH.sub.2—GGFG—COOH 336 peptide NH.sub.2—GFGF—COOH 426 peptide NH.sub.2—GFGGFG—COOH 541 peptide NH.sub.2—GGFGGFGG—COOH 654 peptide NH.sub.2—GGFGGGGFGG—COOH 768

(3) The results for the synthesised monoliths were compared to corresponding results obtained with commercially available DVB solid phase extraction devices—Oasis from the Waters Corporation and Sola from Thermo Fisher Scientific.

(4) FIG. 1 demonstrates the increased binding capacity for the increasing molecular weight analytes on the various synthesised monoliths. The monolith designated “original” was made by the standard process with no secondary porogen. As the molecular weight of the secondary porogen was increased a corresponding increase in binding capacity was observed for the increasing molecular weights of the analytes.

Example 2

(5) Caffeine molecular weight 194, was measured in whole human capillary blood.

(6) A calibration curve was constructed using dilutions of 0.5 mg/mL caffeine stock solution. The following concentrations were chosen: 3.125 μg/mL, 5 μg/mL, 31.25 μg/mL and 62.5 μg/mL. Calibration analysis was carried out by High Pressure Liquid Chromatography (HPLC) under the conditions listed below and gave a correlation of R.sup.2=0.9993 All caffeine concentrations were calculated comparison to the calibration curve obtained.

(7) TABLE-US-00002 LC Conditions Column: 250 × 4.6 mm enable C18G HPLC system: Shimadzu Prominence 20A Flow rate: 1.0 ml/min Mobile phase: 16% acetonitrile with 0.1% TFA Detection: 270 nm Temperature: 25° C. Sample temp: 15° C. Injection volume: 1 μL

(8) Whole blood samples were obtained from two volunteers. The whole blood of the first volunteer was obtained 3 hours after the volunteer had consumed a cup of coffee. The second volunteer had not consumed coffee for the past 24 hours. In each case, 150 μL of blood was lysed with 1350 μL of water. A solid phase extraction (SPE) procedure was conducted according to the following sequence of steps: Precondition cartridge using 2 mL methanol and 2 mL water Load 2 mL of samples Wash with 9 mL water Elute with 1 mL methanol HPLC analysis

(9) A 10 μL sample was injected for HPLC analysis.

(10) The results are shown in FIG. 2.

(11) The caffeine concentration in the blood of volunteer 1 was found to be 2.43 μg/mL. Volunteer 2 was correctly shown to have no measurable caffeine. The healthy level is 1-10 μg/mL. The lethal level is 80 μg/mL.

Example 3

(12) To demonstrate the utility of the hydrophilic surface functionality of a porous polymer monolith with PEGMA surface graft, a 0.5 mg/mL of 3-nitroaniline sample was loaded onto porous polymer monolith 1681 (example 1) and PEGMA grafted monolith 1681. The result showed that sample solution stayed on top of unmodified monolith 1681 but was easily absorbed onto the grafted monolith 1681 without conditioning and equilibration due to the hydrophilic surface allowing the sample to transfer through the polymer.

Example 4

(13) To demonstrate the utility of grafting of the porous polymer monolith to prepare a mixed-mode ion exchange capability, 2-Acrylamido-2-methylpropane sulfonic acid (AMPS) was used as the functional monomer for surface grafting of porous polymer monolith 1681 (example 1). In order to demonstrate the degree of grafting on the mesoporous structure of monolith 1681, amitriptyline (pka=9.4, MW=277.4) was chosen as the target analyte to investigate the binding of different AMPS-grafted 1681 monoliths.

(14) FIG. 3 shows the increased ion exchange functionality of the increased degree of AMPS grafting. The amount of amitriptyline loaded was 100 μg and was completely bound on all samples. The original 1681 is purely hydrophobic and all adsorbed amitriptyline could be eluted with methanol. As the amount of grafted AMPS was increased the amount of amitriptyline that was retained through ionic interactions was increased too. This fraction will not desorb with methanol but requires basic elution conditions. At 3% grafting the binding capacity for amitriptyline is equally shared between the reversed phase and the ionic mode. At 10% grafting and above, the material has turned from a mixed mode sorbent to a true ion exchanger. The high recovery results demonstrate a higher degree of grafting on the mesopores compared to micropores.