Method for Separating Anionic, Cationic, Neutral, and Zwitterionic Arsenic Species

Abstract

A method comprising separating a plurality of arsenic species using a mixed-mode column and a strong acid, the plurality of arsenic species including at least one each of anionic arsenic species, cationic arsenic species, neutral arsenic species, and zwitterionic arsenic species.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of chromatography, including methods for the separation of anionic, cationic, neutral, and zwitterionic arsenic species.

[0002] (Introduction) Chromatography, particularly ion chromatography (IC), is a widely used analytical technique for measuring anionic and cationic analytes in various sample matrices. Analysis of arsenic species by IC-MS typically uses a chromatography column that can operate with either a basic or acidic eluent. When using a basic eluent, neutral and cationic arsenic species are typically poorly retained on the column due to insufficient ionization. When using an acidic eluent, all arsenic species are ionized and can be retained. However, nitric acid, a typical acid chosen, is too strong for the anionic eluent (nitrate ion), resulting in little retention and therefore poor separation of the anionic species. Therefore, improved methods for analyzing arsenic species are needed.

[0003] In "Separation of organic and inorganic arsenic species by HPLC-ICP-MS" (Fresenius J Anal Chem (1999) 363:577-581), Londesborough et al. disclose that separation of several arsenic species is possible using a nitric acid gradient, but that monomethylarsonic acid coelutes with arsenic acid, and trimethylarsine oxide coelutes with tetramethylarsonium ion. Londesborough et al. show that partial separation of trimethylarsine oxide, arsenocholine, and tetramethylarsonium ion is possible with the addition of an eluent modifier.

[0004] Sarzanini et al., in "Metal species determination by ion chromatography" (Trends in analytical chemistry, vol. 20, nos. 6+7, 2001), identified arsenous acid, arsenic acid, monomethylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, tetramethylarsonium ion, arsenobetaine, and arsenocholine. It is shown that this can be obtained by using both an anion exchange column and a cation exchange column. Summary of the Invention

[0005] In a first aspect, a method can include separating a plurality of arsenic species using a mixed-mode column and a strong acid, wherein the plurality of arsenic species includes at least one each of anionic arsenic species, cationic arsenic species, neutral arsenic species, and zwitterionic arsenic species.

[0006] In various embodiments of the first aspect, the anionic arsenic species can be selected from the group consisting of arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid.

[0007] In various embodiments of the first aspect, the neutral arsenic species can include trimethylarsine oxide.

[0008] In various embodiments of the first aspect, the zwitterionic arsenic species can include arsenobetaine.

[0009] In various embodiments of the first aspect, the cationic arsenic species can be selected from the group consisting of arsenocholine and tetramethylarsonium.

[0010] In various embodiments of the first aspect, the mixed-mode column can include a stationary phase having anionic and cationic functional groups.

[0011] In various embodiments of the first aspect, the strong acid can have a pKa of less than 2.0.

[0012] In various embodiments of the first aspect, the strong acid can include methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, chloric acid, or any combination thereof.

[0013] In various embodiments of the first aspect, separating the multiple arsenic species can further use an organic solvent.

[0014] In a second aspect, the method can include separating a plurality of arsenic species using a chromatography column and a strong acid having a counterion, the counterion being NO3 - Contains weaker anion exchange species than

[0015] In various embodiments of the second aspect, the plurality of arsenic species can include anionic arsenic species selected from the group consisting of arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid.

[0016] In various embodiments of the second aspect, the neutral arsenic species can be selected from the group consisting of trimethylarsine oxide.

[0017] In various embodiments of the second aspect, the plurality of arsenic species can include zwitterionic arsenic species. In certain embodiments, the zwitterionic arsenic species can include arsenobetaine.

[0018] In various embodiments of the second aspect, the plurality of arsenic species can include a cationic arsenic species selected from the group consisting of arsenocholine and tetramethylarsonium.

[0019] In various embodiments of the second aspect, the chromatography column may be a mixed-mode column, and the mixed-mode column may include a stationary phase having anionic and cationic functional groups.

[0020] In various embodiments of the second aspect, the strong acid can have a pKa of less than 2.

[0021] In various embodiments of the second aspect, the strong acid can include methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, chloric acid, or any combination thereof.

[0022] In various embodiments of the second aspect, separating the multiple arsenic species can further use an organic solvent.

[0023] In a third aspect, a method can include separating a plurality of arsenic species using a chromatographic column and a strong acid, wherein the plurality of arsenic species includes at least two anionic arsenic species.

[0024] In various embodiments of the third aspect, the anionic arsenic species can be selected from the group consisting of arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid.

[0025] In various embodiments of the third aspect, the plurality of arsenic species can further comprise at least one zwitterionic arsenic species, at least one neutral arsenic species, or at least one cationic arsenic species. In certain embodiments, the neutral arsenic species can comprise dimethylarsinic acid. In certain embodiments, the zwitterionic arsenic can comprise arsenobetaine. In certain embodiments, the cationic arsenic species can be selected from the group consisting of arsenocholine and tetramethylarsonium.

[0026] In various embodiments of the third aspect, the chromatography column can be a mixed-mode column, which can include a stationary phase having anionic and cationic functional groups.

[0027] In various embodiments of the third aspect, the strong acid is NO3 - The anion exchanger may have a counterion that is a weaker anion exchanger than the anion exchanger.

[0028] In various embodiments of the third aspect, the strong acid can have a pKa of less than 2.

[0029] In various embodiments of the third aspect, the strong acid can include methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, chloric acid, or any combination thereof.

[0030] In various embodiments of the third aspect, separating the multiple arsenic species can further use an organic solvent. For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is now made to the following description taken in conjunction with the accompanying drawings and exhibits. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 is a block diagram of an exemplary chromatography system, according to various embodiments. [Figure 2] FIG. 1 is a flow diagram illustrating an exemplary method for analyzing arsenic species in a sample, according to various embodiments. [Figure 3] 1 is a chromatogram showing the separation of various arsenic species. [Figure 4] 1 is a chromatogram showing the separation of various arsenic species. [Figure 5] 1 is a chromatogram showing the separation of various arsenic species.

[0032] It should be understood that the figures are not necessarily drawn to scale, and that objects within the figures are not necessarily drawn to scale in relationship to each other. The figures are representations intended to provide clarity and understanding of various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the figures to refer to the same or like parts. Furthermore, it should be understood that the figures are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION OF THE INVENTION

[0033] Embodiments of methods for the separation of anionic, cationic, neutral, and zwitterionic arsenic species are described herein and in the accompanying presentation.

[0034] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0035] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will understand that these various embodiments may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily understand that the specific order in which the methods are presented and performed is illustrative, and it is contemplated that the order may be changed and still remain within the spirit and scope of the various embodiments disclosed herein.

[0036] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise explained, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong.

[0037] There is an implicit "about" before temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in the present teachings, and thus it is understood that very small, minor deviations fall within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Similarly, the use of "comprises," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0038] As used herein, "a" or "an" can refer to "at least one" or "one or more." Also, the use of "or" is inclusive, so that the phrase "A or B" is true when "A" is true, when "B" is true, or when both "A" and "B" are true. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0039] A "system" refers to a set of components, whether real or abstract, in which each component interacts with or comprises a whole relative to at least one other component within the whole.

[0040] 1 shows an embodiment of a chromatography system 100. The chromatography system 100 may include a pump 102, an electrolytic eluent generator 104, a continuously regenerative trap column 106, a degasser 108, a sample injector 110, a chromatographic separation device 112, an electrolytic suppressor 114, a detector 116, and a microprocessor 118. The chromatographic separation device 112 may be in the form of a capillary column or an analytical column.

[0041] The pump 102 can be configured to pump a liquid from a liquid source 132, such as deionized water, and be fluidly connected to the electrolytic eluent generator 104. The pump 102 can be in the form of a high-pressure liquid chromatography (HPLC) pump.

[0042] An eluent is a liquid containing an acid, a base, a salt, or a mixture thereof that can be used to elute analytes through a chromatography column. Additionally, an eluent can include a mixture of a liquid and a water-miscible organic solvent, where the liquid may include an acid, a base, a salt, or a combination thereof. The electrolytic eluent generator 104 is configured to generate an eluent species. An eluent species refers to a specific acid, base, or salt species that can be added to the eluent. In one embodiment, the eluent species can be a base such as potassium hydroxide, or the eluent species can be an acid such as carbonic acid, phosphoric acid, acetic acid, methanesulfonic acid, or a combination thereof. The eluent can also include an organic solvent such as acetonitrile, methanol, or the like.

[0043] 1, the eluent generator 104 can be configured to receive liquid from the pump 102 and then add eluent species to the liquid. The liquid containing the eluent species can be output from the eluent generator 104 to the inlet of a continuously regenerated trap column 106.

[0044] The continuously regenerated trap column 106 is configured to remove cationic or anionic contaminants from the eluent. The continuously regenerated trap column 106 can include an ion exchange bed with an electrode at the eluent outlet. An ion exchange membrane interface can separate the eluent from a second electrode, and the contaminant ions can sweep through the ion exchange membrane stack toward the second electrode. The ion exchange membrane stack can include one or more ion exchange membranes. In various embodiments, anion removal can utilize an anion exchange bed with a cathode at the eluent outlet separated from the anode by an anion exchange membrane. Alternatively, cation removal can utilize a cation exchange bed with an anode at the eluent outlet separated from the cathode by a cation exchange membrane.

[0045] The degasser 108 may be used to remove residual gases. In one embodiment, the residual gases may be electrolytically generated, such as hydrogen and oxygen. The degasser 108 may include a gas-permeable, liquid-impermeable tubing section, such as an amorphous fluoropolymer, or more specifically, Teflon AF. The flowing liquid may be delivered from the degasser 108 to the sample injector 110 with a substantial portion of the gas removed.

[0046] The sample injector 110 can be used to inject a bolus of liquid sample into the eluent stream. The liquid sample can include multiple chemical components (i.e., matrix components) and one or more analytes of interest. The sample injector 110 can include an autosampler 134, a sample loop 136, and a multiport valve 138. The autosampler 134 can draw the sample from a sample container. The multiport valve 138 can be in a first position that allows the sample to fill the sample loop 136. After the sample loop 136 is filled to a desired level, the multiport valve can be switched to a second position, and the eluent stream can drive the sample onto the chromatographic separation device 112.

[0047] A chromatographic separation device 112 can be used to separate various matrix components present in a liquid sample from analytes of interest. Typically, the chromatographic separation device 112 may be in the form of a hollow cylinder containing a packed stationary phase. As the liquid sample flows through the chromatographic separation device 112, the matrix components and target analytes may have different retention times for elution from the chromatographic separation device 112. Depending on the properties of the target analytes and matrix components, they may have different affinities for the stationary phase of the chromatographic separation device 112. The outlet of the chromatographic separation device 112 may be fluidly connected to an electrolytic suppressor 114.

[0048] The suppressor 114 can be used to reduce eluent conductivity background and improve analyte response by efficiently exchanging eluent counterions for regenerant ions. One type of suppressor is the electrolytic suppressor 114, which can include an anode chamber, a cathode chamber, and an eluent suppression bed chamber separated by an ion exchange membrane. The anode chamber and / or cathode chamber can generate regenerant ions or transport supplied regenerant ions. The eluent suppression bed chamber can include an eluent flow path separated from the regenerant by an ion exchange barrier, and the eluent counterions can exchange with the regenerant ions across the ion exchange barrier. The output of the electrolytic suppressor 114 can be fluidly connected to a detector 116 to measure the presence of separated chemical components in the liquid sample. The suppressor 114 can also have a chemistry that requires a chemical regenerant for operation. Any prior art suppressor with multiple channels configured is suitable for this application.

[0049] The detector 116 may take the form of a UV-visible spectrometer, a fluorescence spectrometer, an atomic fluorescence detector, an atomic emission spectrometer, a refractive index detector, a wireless flow detector, a chiral detector, an electrochemical detector, a conductivity detector, a mass spectrometer, a flame ionization detector, or a combination thereof.

[0050] The electronic circuitry may include a microprocessor 118, a timer, and a memory portion. Additionally, the electronic circuitry may include a power supply configured to apply the respective control signals. The microprocessor 118 may be used to control the operation of the chromatography system 100. The microprocessor 118 may be integrated into the chromatography system 100 or may be part of a personal computer that communicates with the chromatography system 100. The microprocessor 118 may be configured to communicate with and control one or more components of the chromatography system, such as the pump 102, the pump 130, the eluent generator 104, the sample injector 110, and the detector 116. The memory portion may be used to store instructions for setting the magnitude and timing of the current waveform with respect to switching the sample injector 110 to inject a sample.

[0051] FIG. 2 is a flow diagram illustrating a method 200 for analyzing a sample containing arsenic species. At 202, the sample is injected into a chromatography column. Depending on the sample, various pre-injection sample preparation steps can be performed. In various embodiments, a solid or semi-solid sample can be pulverized and suspended in a solution to extract the arsenic species. The liquid solution can be separated from the solid material and injected into the column. In other embodiments, the sample can be a liquid sample, such as a water sample, and can be injected directly into the column without significant sample preparation before injection. The sample can contain multiple arsenic species, including anionic arsenic species, cationic arsenic species, neutral arsenic species, and zwitterionic arsenic species. In various embodiments, the sample can contain at least two anionic arsenic species. In various embodiments, the sample can contain anionic arsenic species and any combination of cationic arsenic species, neutral arsenic species, and zwitterionic arsenic species. For example, the sample can include one or more anionic arsenic species and at least one of a cationic arsenic species, a neutral arsenic species, and a zwitterionic arsenic species. In another example, the sample can include at least one each of anionic arsenic species, cationic arsenic species, a neutral arsenic species, and a zwitterionic arsenic species.

[0052] Anionic arsenic species can behave as anions at all pH levels. Examples of anionic arsenic species include arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid. Neutral arsenic species can behave as cations at low pH levels. Trimethylarsine oxide is an example of a neutral arsenic species. Zwitterionic arsenic species contain both anionic and cationic groups and, when the anionic groups are neutralized, can behave as cations at low pH levels. Arsenobetaine is an example of a zwitterionic arsenic species. Cationic arsenic species can behave as cations at all pH levels. Examples of anionic arsenic species include arsenocholine and tetramethylarsonium.

[0053] The column can include a mixed-mode stationary phase. The mixed-mode stationary phase includes functional groups with two or more different properties, such as cationic functional groups, anionic functional groups, polar groups, etc. In certain embodiments, the mixed-mode stationary phase includes anionic functional groups and cationic functional groups. The arsenic species can be retained on the column by binding to the functional groups.

[0054] At 204, the bound arsenic species can be eluted from the column using an acidic eluent. The acidic eluent can include a strong acid, such as an acid with a pKa of less than 2.0. The strong acid can elute hydronium ions and weak anionic eluent species, such as NO3. - Examples of strong acids with weak anionic eluent species include methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, and chloric acid.

[0055] At 206, the output of the detector can be recorded over time, and at 208, the output of the detector can be used to identify or quantify the arsenic species present in the sample. In certain embodiments, the detector can be an arsenic-selective detector, such as a mass spectrometer. For example, method 200 can be performed by an ion chromatography mass spectrometer (IC-MS).

[0056] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. Rather, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0057] Furthermore, in describing various embodiments, the specification may present methods and / or processes in terms of a particular order. However, to the extent that the method or process does not rely on the particular order of steps described herein, the method or process should not be limited to the particular order of steps described. As one of ordinary skill in the art will understand, other orders of steps may be possible. Thus, the particular order of steps described herein should not be construed as a limitation on the claims. In addition, claims directed to the method and / or process should not be limited to performing those steps in the order written; one of ordinary skill in the art will readily understand that the order may be changed and still remain within the spirit and scope of the various embodiments.

[0058] Any of the operations forming part of the embodiments described herein are useful machine operations. The embodiments described herein also refer to devices or apparatus for performing those operations. The systems and methods described herein may be specially constructed for the required purposes, or they may be general-purpose computers selectively activated or configured by a computer program stored in the computer. In particular, various general-purpose machines may be used with computer programs written in accordance with the teachings herein, or it may be convenient to construct more specialized apparatus to perform the required operations.

[0059] Example Figure 3 is a chromatogram showing the separation of a sample containing nine arsenic species. The separation uses an IonPac AS7 column with methanesulfonic acid as the eluent. From 0 to 5 minutes, the methanesulfonic acid is at a concentration of 2.0 mM, increasing to 20 mM from 5 to 10 minutes, and then maintained at 20 mM from 10 to 25 minutes. The sample contains (1) arsenous acid, (2) methylarsonic acid, (3) dimethylarsinic acid, (4) phenylarsinic acid, (5) arsenic acid, (6) arsenobetaine, (7) thyme thyrsine oxide, (8) arsenocholine, and (9) tetramethylarsonium.

[0060] Figure 4 shows a chromatogram showing the separation of a sample containing 17 arsenic species. The separation was performed using an IonPac AS7 column with methanesulfonic acid as the eluent. From 0 to 5 minutes, the methanesulfonic acid was at a concentration of 2.0 mM, increased to 20 mM from 5 to 10 minutes, and then maintained at 20 mM from 10 to 25 minutes. The samples included (1) arsenous acid, (2) phenylarsine oxide, (3) methylarsonic acid, (4) dimethylarsinic acid, (5) phenylarsinic acid, (6) 2-nitrophenylarsinic acid, (7) 2-aminophenylarsinic acid, (8) 4-hydroxyphenylarsinic acid, (9) p-arsanilic acid, (10) carbasone, (11) arsenic acid, (12) arsenobetaine, (13) nitrosone, (14) roxyarsone, (15) timetylarsine oxide, (16) arsenocholine, and (17) tetramethylarsonium.

[0061] Figure 5 is a chromatogram showing the separation of a sample containing nine arsenic species. The separation uses an IonPac CS5A column with methanesulfonic acid as the eluent. From 0 to 2 minutes, the methanesulfonic acid is at a concentration of 2.0 mM, increasing to 6 mM from 2 to 8 minutes, and then to 80 mM from 8 to 20 minutes. The sample contains (1) arsenous acid, (2) methylarsonic acid, (3) phenylarsinic acid, (4) arsenic acid, (5) dimethylarsinic acid, (6) arsenobetaine, (7) thymethiol oxide, (8) arsenocholine, and (9) tetramethylarsonium.

Claims

1. 1. A method comprising:

1. A method comprising: separating a plurality of arsenic species using a mixed-mode column and a strong acid, wherein the plurality of arsenic species comprises at least one each of anionic arsenic species, cationic arsenic species, neutral arsenic species, and zwitterionic arsenic species.

2. 2. The method of claim 1, wherein the anionic arsenic species is selected from the group consisting of arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid.

3. 10. The method of claim 1, wherein the neutral arsenic species comprises trimethylarsine oxide.

4. 10. The method of claim 1, wherein the zwitterionic arsenic species comprises arsenobetaine.

5. 2. The method of claim 1, wherein the cationic arsenic species is selected from the group consisting of arsenocholine and tetramethylarsonium.

6. 10. The method of claim 1, wherein the mixed-mode column comprises a stationary phase having anionic and cationic functional groups.

7. The strong acid has a pK of less than 2.0 a 2. The method of claim 1, comprising:

8. 10. The method of claim 1, wherein the strong acid comprises methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, chloric acid, or any combination thereof.

9. 10. The method of claim 1, wherein separating the arsenic species further uses an organic solvent.

10. 1. A method comprising: A chromatography column and a NO 3 - and a strong acid having a counterion comprising an anion exchange species weaker than

11. 11. The method of claim 10, wherein the plurality of arsenic species comprises anionic arsenic species selected from the group consisting of arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid.

12. 11. The method of claim 10, wherein the neutral arsenic species is selected from the group consisting of trimethylarsine oxides.

13. 11. The method of claim 10, wherein the plurality of arsenic species comprises zwitterionic arsenic species.

14. 14. The method of claim 13, wherein the zwitterionic arsenic species comprises arsenobetaine.

15. 11. The method of claim 10, wherein the plurality of arsenic species comprises a cationic arsenic species selected from the group consisting of arsenocholine and tetramethylarsonium.

16. 11. The method of claim 10, wherein the chromatography column is a mixed-mode column, the mixed-mode column comprising a stationary phase having anionic and cationic functional groups.

17. The strong acid has a pK of less than 2 a 11. The method of claim 10, comprising:

18. 11. The method of claim 10, wherein the strong acid comprises methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, chloric acid, or any combination thereof.

19. 11. The method of claim 10, wherein separating the arsenic species further uses an organic solvent.

20. 1. A method comprising: separating the plurality of arsenic species, the plurality comprising at least two anionic arsenic species, using a chromatographic column and a strong acid.

21. 21. The method of claim 20, wherein the anionic arsenic species is selected from the group consisting of arsenous acid, methylarsonic acid, dimethylarsinic acid, phenylarsinic acid, and arsenic acid.

22. 21. The method of claim 20, wherein the plurality of arsenic species further comprises at least one zwitterionic arsenic species, at least one neutral arsenic species, or at least one cationic arsenic species.

23. 23. The method of claim 22, wherein the neutral arsenic species comprises dimethylarsinic acid.

24. 23. The method of claim 22, wherein the zwitterionic arsenic comprises arsenobetaine.

25. 23. The method of claim 22, wherein the cationic arsenic species is selected from the group consisting of arsenocholine and tetramethylarsonium.

26. 21. The method of claim 20, wherein the chromatography column is a mixed-mode column, the mixed-mode column comprising a stationary phase having anionic and cationic functional groups.

27. The strong acid is NO 3 - 21. The method of claim 20, wherein the anion exchanger has a counterion that is a weaker anion exchange species than the anion exchanger.

28. The strong acid has a pK of less than 2 a 21. The method of claim 20, comprising:

29. 21. The method of claim 20, wherein the strong acid comprises methanesulfonic acid, ethanesulfonic acid, hydrochloric acid, hydrobromic acid, iodic acid, chloric acid, or any combination thereof.

30. 21. The method of claim 20, wherein separating the arsenic species further uses an organic solvent.

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