Millifluid chromatography plate

Abstract

Chromatographic plates for the separation and detection of ion-selective ligands and complexes thereof. A combination of an enhanced chromatographic separation method and an ultraviolet imaging device for detection has been developed for selective and simultaneous multi-ion sensing. The methods involve making microfluidic channels on a porous chromatography substrate to enhance the separation of ligands comprising ultraviolet chromophores and complexes thereof with ions of interest. The chromatography plate is analyzed under an ultraviolet imaging device to quantify the concentrations of the different ions present in the sample.

Description

Technical Field

[0001] The invention relates to a chromatographic plate for separation and detection of ion selective ligands and complexes thereof. Background Art

[0002] Compound separation is crucial in analyzing chemical compounds as pure compounds provide reliable results without interference from other compounds, making chromatographic separation a useful tool in analytical chemistry. Chromatographic separation can be in the form of a simple thin layer chromatography plate to quantitatively determine the amount of compounds in a solution or in the form of a complex and efficient chromatography system that can be coupled to different detectors that can qualitatively and quantitatively determine different compounds in a solution. The differentiating factors lie in the separation method and the detection mode of the separated compounds. Separation is the core of chromatographic methods and the principle behind compound separation revolves around the different interactions between the compounds present in the sample and the stationary and mobile phases of the chromatographic system. Detection, on the other hand, is crucial for the effectiveness and efficiency of analytical separation.

[0003] Ion sensing can be achieved using high-performance chromatography systems equipped with specialized separation columns coupled to charge detectors or mass spectrometers. However, this approach requires samples to be sent to a central laboratory with sufficient equipment. Although high-performance liquid chromatography coupled to a mass spectrometer can separate and detect ions very accurately, it is not feasible in situations where rapid analysis and results are required, laboratories that do not have access to such equipment, and the cost of the analysis needs to be affordable.

[0004] Therefore, there is a need to develop an affordable and rapid chromatographic separation method for separating and detecting ions that are less sensitive to interference from other ions.

[0005] It is therefore an object of the present invention to provide an enhanced chromatography plate for separation and detection of ion-selective ligands and their complexes which solves the above-mentioned disadvantages. Summary of the invention

[0006] In one aspect of the present invention, there is provided a chromatography plate, comprising:

[0007] Porous substrates;

[0008] at least one channel on the substrate, wherein the channel further comprises:

[0009] a sample well for deposition of solution;

[0010] a ligand well for depositing ligands;

[0011] a mixing zone to allow mixing and interaction of the solution with the ligand;

[0012] an elongated separation region for separating a ligand and a complex formed with the ligand; and

[0013] an effluent well, in which excess solution from the channel is collected;

[0014] Characterized in that the channels are millifluidic and the deposited ligands selectively form complexes with ions found in solution, and the mixture of ligands and their complexes are then separated along separation zones based on their interaction forces with the substrate.

[0015] Advantageously, the millifluidic channels provide an enhanced chromatography plate that enables equipment-free chromatographic separation of ligands containing UV chromophores and their complexes, compared to prior art techniques disclosing nanostructured channels, without the need for expensive equipment for imaging to view the deposition of ligands of separated compounds within the channel.

[0016] Advantageously, the channels confine the flow of the solution, eliminating uneven flow patterns, enhancing the capillary action of the solution, which in turn enhances the separation of ions found in the solution.

[0017] Advantageously, spiral channels can be used to increase the number of ligands that can be separated with good resolution, since straight channels are limited by the length of the chromatography plate.

[0018] Preferably, the porous substrate is silica or cellulose.

[0019] In one embodiment, the depth of the channel is in the range of 10 to 20 μm, and the width of the channel is in the range of 1.5 mm to 3.0 mm.

[0020] In one embodiment, the mixing region includes at least two bends to enable adequate mixing of the solution and the ligand.

[0021] In one embodiment, the ligand comprises a UV chromophore.

[0022] In one embodiment, the ligand having an ultraviolet chromophore is selected from 4'-aminobenzo-15-crown 5-ether, 4'-aminobenzo-18-crown 6-ether, diamino-benzo-9-crown-3, 4'-aminobenzo-24-crown-8, 5,6-benzo-4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacos-5-ene to form complexes with cadmium, potassium, beryllium, cesium, lead and radium ions, respectively.

[0023] In one embodiment, the ligand having an ultraviolet chromophore is selected from N,N'-dibenzyl-4,13-diaza-18-crown-6-ether, aza-15-crown-5, 7,16-dibenzyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane to form a complex with the lead ion.

[0024] In one embodiment, the ligand having an ultraviolet chromophore is selected from iron and 6-thioguanine complex, iron and 6-amino-2-mercaptobenzothiazole complex, iron and 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate to form a complex with arsenic ions.

[0025] In one embodiment, a single channel is fabricated to allow deposition of multiple ligands that are selective for different ions found in solution, thereby enabling simultaneous separation of the ligands and the complexes formed.

[0026] Advantageously, chromatography plates can help enhance chromatographic separations by improving uniformity and resolution and allow for equipment-free separation of ligands and their complexes.

[0027] In one embodiment, after separation, detection is performed using a UV imaging device to quantify the concentrations of the different ions present in the solution.

[0028] In one aspect of the present invention, there is provided a method for producing a chromatography plate, comprising the steps of:

[0029] coating a chromatography plate including a substrate with a UV-curable polymer mixture;

[0030] The coated panels were placed in a vacuum chamber to remove any air bubbles;

[0031] positioning the coated plate beneath a liquid crystal display mask so that at least one channel design can be transferred to the coated plate when ultraviolet light is utilized to cure the polymer mixture;

[0032] washing away the uncured polymer mixture and allowing the plate to dry; and

[0033] selectively depositing ligands for the ions of interest in channels formed in the plate;

[0034] It is characterized in that the channels are millifluidic and are made to allow the deposition of multiple ligands selective for different ions found in solution, thus enabling the simultaneous separation of the ligands and the complexes formed with the ligands.

[0035] In one aspect of the present invention, there is provided the use of a chromatography plate for separation and detection of ion-selective ligands and their complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] It will be convenient to further describe the invention with respect to the accompanying drawings which illustrate possible arrangements of the invention. Other arrangements of the invention are possible, and consequently the particularity of the accompanying drawings should not be understood as superseding the generality of the preceding description of the invention.

[0037] Figure 1 A millifluid chromatography plate is shown.

[0038] Figure 2 Shown is the separation of ligands and their complexes on a straight channel millifluid chromatography plate.

[0039] Figure 3 Shown is the separation of ligands and their complexes on a spiral channel millifluid chromatography plate.

[0040] Figure 4 A method for producing a millifluid chromatography plate is shown. DETAILED DESCRIPTION

[0041] The separation component of the present invention is intended to enhance preferably commercially available silica or cellulose chromatography plates to produce millifluid chromatography plates that can be used to separate multiple ion-selective ligands and their complexes simultaneously with good resolution.

[0042] Figure 1 A millifluid chromatography plate of the present invention is shown. The plate comprises a porous substrate (102) and at least one millifluid channel (104) on the substrate, wherein the porous substrate is preferably silica or cellulose. The channel further comprises a sample well (106) for depositing a solution, a ligand well (108) for depositing a ligand, a mixing region (110) for allowing the solution and the ligand to mix and interact, an elongated separation region (112) for separating the ligand and the complex formed of the ligand, and an effluent well (114) for collecting excess solution from the channel. The deposited ligand selectively forms a complex with the ions in the solution, and then the mixture of the ligand and its complex is separated along the separation region based on their interaction forces with the substrate.

[0043] In addition to the thickness of the substrate, the depth of the millifluidic channel is in the range of 10-20 μm, while the width of the channel is in the range of 1.5 mm to 3.0 mm. The mixing region comprises at least two bends, wherein the angle of the bends is 20° to 70°, preferably 60°. The bends allow the solution to mix thoroughly with the ligand without restricting the flow of the solution and increasing the time required for the ligand to separate from its complex. The ligand includes an ultraviolet chromophore to enable detection of the separated ligand and its complex using an ultraviolet imaging device. The volume of the deposited ligand depends on the ion concentration that the ultraviolet imaging device is designed to detect.

[0044] Figure 2The separation of ligands and their complexes on a straight channel millifluid chromatography plate is shown. Specifically, Figure 2 a shows the separation of the ligand using a blank sample (deionized water), compared to Figure 2 b shows the separation of ligands and their complexes using a sample solution containing ions on a millifluidic chromatography plate with a straight channel. The straight channel may be limited by the length of the chromatography plate.

[0045] Figure 3 The separation of ligands and their complexes on a spiral channel millifluid chromatography plate is shown. Specifically, Figure 3 a shows the separation of the ligand using a blank sample (deionized water), compared to Figure 3 b shows the separation of ligands and their complexes using a sample solution containing ions on a millifluid chromatography plate with a spiral channel. The spiral channel can be used to increase the number of ligands that can be separated with good resolution.

[0046] When a drop of solution containing the ions of interest is deposited onto the sample well, the ligands will selectively bind to the corresponding ions for which they are selective, and a mixture of ligands and complexes will flow along the millifluidic channel. After the separation on the chromatographic plate is complete, the plate can be inserted into a UV imaging device, and the UV image is captured and analyzed using proprietary software to provide quantitative results of the ions of interest present in the analyzed solution. The detection assembly of the present invention is based on the use of ligands with UV chromophores that can absorb UV light. When the millifluidic chromatographic plate is used to separate ligands and their complexes, they will appear as black spots at different positions on the UV imaging device (after irradiating the substrate under UV light) relative to the intensity of their interaction forces with the porous substrate; the more polar groups present on the modified ligand or its complex, the higher its interaction force with the substrate, and the slower its flow along the millifluidic channel. The intensity of the black spot will correspond to the concentration of the ligand and / or its complex in that spot. In order to identify the ligand and its complex, the chromatographic plate must first be calibrated by separating the ligand using deionized water. The relative distance from the starting point of the deposited ligand-chromophore mixture shows the location of the ligand. When solutions other than deionized water are used, any additional points formed will correspond to the respective complexes of the ligand ( Figure 2 b and Figure 3 b) When a fixed amount of fluid sample is deposited on the sample well, the location of the spots should be similar and this information can be stored in the proprietary sensor system and software during calibration.

[0047] Millifluidic channels confine the flow of solutions within the channel, eliminating uneven flow patterns, enhancing the capillary action of the solution and thus the separation of ions in solution, and a single millifluidic channel is fabricated to allow the deposition of multiple ligands that are selective for different ions found in solution, enabling the simultaneous separation of ions.

[0048] The microfluidic chromatography plates of the present invention can help enhance chromatographic separations by improving uniformity and resolution, and allow for equipment-free separation of ligands and their complexes.

[0049] After separation, detection is performed using a UV imaging device to quantify the concentration of the different ions present in the solution.

[0050] Figure 4 A method for producing millifluid chromatography plates is shown. A UV-curable polymer mixture is prepared by mixing polymer, crosslinker and photoinitiator in a solvent (depending on the polymer) in a ratio that yields a solution with a viscosity of 500 to 1000 cP (Table 1) to allow the polymer mixture to flow easily into the porous matrix of a commercial silica or cellulose chromatography plate.

[0051] Table 1 lists possible component combinations and recommended mixing ratios for preparing UV-curable polymer mixtures.

[0052]

[0053] Table 1

[0054] The prepared polymer mixture is then deposited on a porous substrate, ensuring that the entire substrate is covered (202). The coated substrate is placed in a vacuum chamber to force any bubbles out of the porous substrate and the polymer mixture, thereby producing a uniform, bubble-free coating (204). The coated substrate is then placed under a liquid crystal display mask to enable at least one microfluidic channel design to be transferred to the coated plate when ultraviolet light is used to cure the polymer mixture (206). The chromatographic plate is washed in the same solvent previously used to prepare the polymer mixture to remove the uncured polymer mixture (208) and then dried in an oven at a temperature 10°C above the boiling point of the solvent used to prepare the polymer mixture for 30 minutes. Selected ligands having ultraviolet chromophores specific to the ions of interest are dissolved using low boiling point solvents (such as acetone, ethanol, isopropanol or acetonitrile) (Table 2) and deposited at the beginning of the millifluidic channel. The amount of deposited ligand depends on the desired sensitivity and detection range. Millifluidic chromatography plates are used to analyze solutions for ions of interest by depositing a fixed amount (50-100 μL) of solution onto the sample well of a millifluidic channel, allowing ligands and complexes to separate along the millifluidic channel.

[0055] Table 2 lists the ligands with UV chromophores and the corresponding ions of interest for which they are selective.

[0056]

[0057] Table 2

[0058] The present invention provides an enhanced chromatography plate that enables equipment-free chromatographic separation of ligands and their complexes containing UV chromophores. When coupled with a UV imaging device, the present invention is capable of simultaneous separation and detection of multiple ions in an aqueous environment. The present invention can be used as an in-situ ion sensor and has great commercial value in, for example, the water quality monitoring and point-of-care device markets.

[0059] It should be understood by those skilled in the art that the present invention may also include further additional modifications made to the chromatography plate without affecting the overall function of the chromatography plate.

Claims

1. A chromatography plate, comprising: A porous substrate (102); as well as at least one channel (104) on the substrate, wherein the channel further comprises: a sample well (106) for depositing a solution; a ligand well (108) for depositing ligands; a mixing zone (110) which allows the solution and the ligand to mix and interact; an elongated separation region (112) for separating the ligand and the complex formed with the ligand; and an effluent well (114) in which excess solution from the channel is collected; Characterized in that the channels are millifluidic and the deposited ligands selectively form complexes with ions found in solution, and the mixture of ligands and their complexes are then separated along separation zones based on their interaction forces with the substrate.

2. The chromatographic plate according to claim 1, wherein The porous substrate is preferably silica or cellulose.

3. The chromatographic plate according to claim 1, wherein The depth of the channels is in the range of 10 to 20 μm, while the width of the channels is in the range of 1.5 mm to 3.0 mm.

4. The chromatographic plate according to claim 1, wherein The mixing region includes at least two bends to allow for thorough mixing of the solution and the ligand.

5. The chromatographic plate according to claim 1, wherein The ligand includes a UV chromophore.

6. The chromatographic plate according to claim 5, wherein The ligand having an ultraviolet chromophore is selected from 4'-aminobenzo-15-crown 5-ether, 4'-aminobenzo-18-crown 6-ether, diamino-benzo-9-crown-3, 4'-aminobenzo-24-crown-8, 5,6-benzo-4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacos-5-ene to form complexes with cadmium, potassium, beryllium, cesium, lead and radium ions, respectively.

7. The chromatography plate according to claim 5, wherein The ligand having an ultraviolet chromophore is selected from N,N'-dibenzyl-4,13-diaza-18-crown-6-ether, aza-15-crown-5,7,16-dibenzyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane to form a complex with the lead ion.

8. The chromatography plate according to claim 5, wherein The ligand having an ultraviolet chromophore is selected from a complex of iron and 6-thioguanine, a complex of iron and 6-amino-2-mercaptobenzothiazole, and a complex of iron and 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate to form a complex with arsenic ions.

9. The chromatography plate according to claim 1, wherein A single channel is fabricated to allow deposition of multiple ligands selective for different ions found in solution, enabling simultaneous separation of the ligands and the complexes formed.

10. The chromatography plate according to claim 1, wherein After separation, detection is performed using a UV imaging device to quantify the concentration of the different ions present in the solution.

11. A method for producing a chromatography plate according to claim 1, comprising the following steps: coating a chromatography plate (202) comprising a substrate with a UV-curable polymer mixture; placing the coated panel in a vacuum chamber to remove any air bubbles (204); positioning the coated plate beneath a liquid crystal display mask so that when ultraviolet light is utilized to cure the polymer mixture, at least one channel design can be transferred to the coated plate (206); washing away the uncured polymer mixture and drying the sheet (208); as well as Depositing ligands selective for the ions of interest in channels formed in the plate; Characteristically, the channels are millifluidic and are fabricated to allow the deposition of multiple ligands selective for different ions found in solution, so as to enable the simultaneous separation of the ligands and the complexes formed of said ligands.

12. Use of the chromatography plate according to claim 1 for separation and detection of ion-selective ligands and their complexes.

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