Extraction separation using magnetic beads

Abstract

A method for purifying a substance in a solution in a simple streamlined process using a magnetic porous particle. For easy small scale purification of a substance, the magnetic porous particle is coated with either a hydrophilic or hydrophobic liquid and transferred into a second liquid containing the substance under conditions which allow said substance to partition into the first liquid within said magnetic porous particle. Finally the magnetic porous particle is removed from said second liquid, wherein the first and second liquid are substantially immiscible and the partition coefficient P of the substance between the first and second liquid is greater than 1.

Claims

1. A method of extracting a substance from a liquid, the method comprising; contacting a porous particle comprising a superparamagnetic or ferromagnetic material with a first liquid; transferring said porous particle to a second liquid containing a substance under conditions to allow said substance to partition into said first liquid within said porous particle; and separating the porous particle from said second liquid, wherein the first and second liquid are substantially immiscible and the partition coefficient P of the substance between the first and second liquid is greater than 1.

2. The method according to claim 1, wherein the porous particle comprises a plurality of pores each having a pore surface.

3. The method according to claim 2, wherein said pore surface has a higher affinity for the first liquid than for the second liquid.

4. The method according to claim 1, wherein said superparamagnetic material is coated with an inert synthetic polymer.

5. The method according to claim 1, wherein the superparamagnetic material is ferric oxide.

6. The method according to claim 1, wherein the porous particle comprises a natural or a synthetic hydrophilic polymer.

7. The method according to claim 6, wherein said hydrophilic polymer is selected from the group consisting of agarose, dextran, cellulose, polyvinyl alcohol, polyacrylamide, hydrophilized polymers and silica glass.

8. The method according to claim 1, wherein the porous particle comprises a natural or a synthetic hydrophobic polymer.

9. The method according to claim 8, wherein said hydrophobic polymer is selected from the group consisting of polyvinyl benzene, polymethacrylate, polystyrene, polypropene and polythene.

10. The method according to claim 8, wherein the first liquid is hydrophobic and the second liquid is hydrophilic.

11. The method according to claim 1, wherein the porous particle comprises a surface coated or coupled or functionalized with a hydrophobic agent.

12. The method according to claim 11, wherein said hydrophobic agent is selected from the group consisting of a hydrocarbon group, an aliphatic group and an aromatic group.

13. The method according to claim 1, wherein the porous particle comprises a surface coated or coupled or functionalized with a hydrophilic agent.

14. The method according to claim 13, wherein said hydrophilic agent is selected from the group consisting of a polysaccharide, agarose, dextran, cellulose, sugar, sorbitol and manitol.

15. The method according to claim 1, wherein the first liquid is hydrophilic and the second liquid is hydrophobic.

16. The method according to claim 1, wherein the mean particle diameter of the porous particle is in the range of 5 m to 1000 m.

17. The method according to claim 1, wherein the mean particle diameter of the porous particle is in the range of 30 m to 200 m.

18. The method according to claim 1, wherein the volume of the second liquid is selected from the group in the range of 1 l to 10 l, 10 l to 100 l, 100 l to 1 ml, 1 ml to 1 L and 1 L to 10 L.

19. The method according to claim 1, wherein the method of separating the porous particle from said second liquid is by magnetic separation.

20. The method according to claim 1, wherein the method of separating the porous particle from said second liquid is by centrifugation.

21. The method according to claim 1, wherein the method of separating the porous particle from said second liquid by filtration.

22. The method according to claim 1, wherein the method is for use in separating, purifying or concentrating a substance which is present alone or in a mixture in a liquid.

23. The method according to claim 1, wherein the substance is a synthetic compound.

24. The method according to claim 1, wherein the substance is an organic molecule and/or biological molecule.

25. The method according to claim 1, wherein the substance is selected from the group consisting of lipid, nucleic acid, protein, glycoprotein, glycopeptide, phosphoprotein, phosphopeptide and carbohydrate.

26. The method according to claim 1 wherein the substance is selected from the group consisting of agrochemical, pesticide, plasticizer, drug, cosmetic, dye and environmental pollutant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 presents the results of the Magnetic Sepharose N6 beads after the addition of HCl and after the subsequent addition of NaOH.

DETAILED DESCRIPTION

Definitions

(2) To more clearly and concisely described and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following description and the appended claims. Throughout the specification, exemplification of specific terms should be considered as non-limiting examples.

(3) The term immiscible as used herein will mean a liquid substance that in some proportion doesn't form a homogenous solution.

(4) As used herein, the term partition coefficient will mean the equilibrium concentration of a substance in liquid 1 divided by the equilibrium concentration of a substance in liquid 2.

Chemicals and Materials Used

(5) A list of the chemicals and their sources is given below:

(6) 2,6-dichloroindophenol (Sigma, no D1878-5G) 0.5 mg/ml in 20 mM NaOH

(7) Chloroform (Sigma, no 288306-100 ml)

(8) 0.2M HCL (Merck 1.00317.1000)

(9) 0.5M NaOH (Merck, 1.006460.1000)

(10) 1.5 ml Eppendorf tube (Axygen, no 311-08-081)

(11) MagRack 6 (GE Healthcare UK Ltd. Product code 28-9489-64)

(12) SpectraMax Spectrophotometer (Molecular Devices)

Experimental Description and Results

Production of Magnetic Sepharose Beads

(13) The Magnetic Sepharose N6 beads were produced using the protocol described in U.S. Pat. No. 7,897,257 (Alterman et al.). The magnetic metal particles were treated with an amphiphilic agent, after which a polymerizable monomer and a radical initiator were added. The temperature was increased to cause the polymerization of the monomer. The polymer-coated magnetic particles are then emulsified into agarose.

(14) The Magnetic Sepharose C8 beads were produced by further reacting the Magnetic Sepharose N6 beads with allyl glycidyl ether and then substituted with Octyl-thiol.

Hydrophilic Magnetic Sepharose N6 Beads Experimental Method

(15) The dye, 2,6-dichloroindophenol, used in the experiment is blue and water soluble at a neutral to basic pH. At acidic pH it is protonated, red in colour and organic soluble.

(16) 100 l of magnetic slurry yields 22 l of drained beads, 100 l of the slurry was added to microtubes and the supernatant (20% EtOH) was removed. The beads were washed twice with 500 l dH.sub.2O.

(17) The Magnetic Sepharose N6 Beads (Mag-N6) were equilibrated twice with 1 ml of dH2O in order to saturate the beads with the solution. MagRack 6 was used to attract the magnetic beads to the base of the microtube and the water was removed. 1 ml of chloroform was added to Mag-N6 (saturated with water) and 50 l of 2,6-dichloroindophenol (0.5 mg/ml) was added to the tube. The mixture was acidified by the addition of 20 l 0.2 M HCl. pH was thereafter adjusted to basic by the addition of 8 l 0.5M NaOH.

Hydrophobic Magnetic Sepharose C8 Beads Experimental Method

(18) The dye, 2,6-dichloroindophenol, used in the experiment is blue and water soluble at a neutral to basic pH. At acidic pH it is protonated, red in colour and organic soluble.

(19) 100 l of magnetic slurry yields 22 l of drained beads, 100 l of the slurry was added to microtubes and the supernatant (20% EtOH) was removed. The beads were washed twice with 500 l dH.sub.2O.

(20) The Magnetic Sepharose C8 Beads (Mag-C8) was equilibrated twice with 1 ml of chloroform in order to saturate the beads with the solution. MagRack 6 was used to attract the magnetic beads to the base of the microtube and the chloroform was removed. 1 ml of water was added to Mag-C8 (saturated with chloroform) and 50 l of 2,6-dichloroindophenol (0.5 mg/ml) was added to the tube. The mixture was acidified by the addition of 20 l 0.2 M HCl. Using the MagRack 6 the acidic solution was replaced with 20 mM NaOH.

Results

Magnetic Sepharose N6 Beads Results

(21) After the addition of 2,6-dichloroindophenol to the chloroform solution, the blue dye immediately localized into the hydrophilic water saturated Mag-N6 beads.

(22) The addition of 20 l of 0.2 M HCl caused a protonation of the dye and the delocalisation of the dye from the beads to the surrounding chloroform phase indicated by the colour red. This process was reversed by the addition of 8 l of 0.5M NaOH to the chloroform, deprotonating 2,6-dichloroindophenol. The dye turned blue and water soluble and returned into the beads leaving a colourless chloroform solution. The results are presented graphically in FIG. 1.

(23) FIG. 1 shows the results after the addition of HCl (lane 1) and after the subsequent addition of NaOH (lane 2).

Magnetic Sepharose C8 Hydrophobic Beads Results

(24) 2,6-dichloroindophenol indicator was added to the tube containing the chloroform saturated Mag-C8 beads, the indicator was blue as it remained in the water. 20 l of 0.2M HCL was added to the Mag-C8 beads and the water phase containing the indicator turned red indicating that the dye was protonated.

(25) The beads were subsequently vortexed and the water turned slightly pink because the indicator was localised into the chloroform saturated Mag-C8 bead. The water phase was removed and replaced with 20 mM NaOH and water. The beads were vortexed and the indicator in its deprotonated blue form was delocalized back into the water phase. The absorbance was measured at 590 nm, which is the peak absorbance for the deprotonated blue form of the 2,6-dichloroindophenol indicator. It was calculated that 84% of the dye had been extracted into the chloroform saturated Mag-C8 beads.

(26) While illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practised by other than the described embodiments, which are presented for the purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.