阅读说明：本技术 用于从样本中提取分析物的系统和方法 (System and method for extracting an analyte from a sample ) 是由 A·R·科马雷克 R·吉亚内蒂 M·D·科马雷克 罗纳尔德·J·科马雷克 赖安·J·科马雷克 于 2020-04-29 设计创作，主要内容包括：用于从样本中提取分析物的系统和方法。该系统包括用于接收样本和反应溶液的反应器皿、用于将样本与反应溶液混合的混合器、过滤器以及用于使来自反应混合物的可溶性组分(包括溶解的分析物)从反应器皿通过的排放部。纯化器皿位于反应器皿下方。选择性吸附剂设置在纯化器皿中,用于保留来自于自反应混合物的可溶性组分的污染物并使纯化的分析物通过。蒸发容器位于纯化器皿下方。加热器加热蒸发室并从纯化的分析物中蒸发溶剂,该分析物然后可定量测量。(Systems and methods for extracting an analyte from a sample. The system includes a reaction vessel for receiving the sample and the reaction solution, a mixer for mixing the sample with the reaction solution, a filter, and a drain for passing soluble components (including dissolved analyte) from the reaction mixture through the reaction vessel. A purification vessel is located below the reaction vessel. A selective adsorbent is disposed in the purification vessel for retaining contaminants from soluble components of the reaction mixture and passing purified analytes therethrough. The evaporation vessel is located below the purification vessel. The heater heats the evaporation chamber and evaporates the solvent from the purified analyte, which can then be quantitatively measured.)

1. A system for extracting an analyte from a sample, the system comprising:

a reaction chamber comprising a reaction vessel comprising

A reaction vessel column for receiving the sample and a reaction solution,

a mixer at a bottom end of the reaction vessel for mixing the sample with the reaction solution,

a reaction vessel filter located at a bottom end of the reaction vessel for retaining insoluble components from a reaction mixture of the sample and the reaction solution and passing soluble components from the reaction mixture, and

a reaction vessel discharge located at a bottom end of the reaction vessel and below the reaction vessel filter for discharging a soluble component from the reaction mixture from the reaction vessel, the soluble component comprising a dissolved analyte;

a purification chamber located below the reaction chamber, the purification chamber comprising a purification vessel comprising

A purification vessel column to receive a soluble component from the reaction mixture from the reaction vessel discharge, the soluble component including the dissolved analyte,

a selective adsorbent disposed in the purification vessel for retaining contaminants from soluble components from the reaction mixture and passing purified analytes, an

A purification vessel discharge located at a bottom end of the purification vessel and below the selective adsorbent, the purification vessel discharge for discharging the purified analyte from the purification vessel;

an evaporation chamber located below the purification chamber, the evaporation chamber comprising an evaporation container for receiving the purified analyte from the purification vessel discharge; and

a heater for heating the evaporation chamber and evaporating solvent from the purified analyte.

2. The system of claim 1, wherein the reaction vessel, the purification vessel, and the evaporation vessel are vertically aligned along the same axis.

3. The system of claim 1, wherein the reaction vessel is removable.

4. The system of claim 1, wherein the reaction vessel further comprises a reaction vessel base located at the bottom end of the reaction vessel and below the reaction vessel filter.

5. The system of claim 4, wherein the mixer comprises a mixing paddle above the reaction vessel filter, and wherein the system further comprises a magnetic mixer drive below the reaction vessel base, the magnetic mixer drive comprising an electrical winding configured to generate an alternating magnetic flux field for driving the mixing paddle about an axis of rotation.

6. The system of claim 1, wherein the purification vessel is removable.

7. The system of claim 1, wherein the reaction vessel further comprises a reactor vessel closure at a top end of the reaction vessel, wherein the reactor vessel closure comprises a reactor vessel closure orifice.

8. The system of claim 1, wherein the purification vessel further comprises a diffuser for diffusing a soluble component from the reaction mixture received from the reaction vessel discharge, the soluble component comprising the dissolved analyte, wherein the diffuser is located at a top end of the purification vessel.

9. The system of claim 1, wherein the selective adsorbent is a solid phase filter material.

10. The system of claim 9, wherein the solid phase filter material is selected from the group consisting of siliceous earth or diatomaceous earth.

11. The system of claim 1, wherein the purification vessel further comprises a purification vessel filter at a bottom end of the purification vessel for retaining the selective adsorbent and passing the purified analyte.

12. The system of claim 1, further comprising a blower for blowing air heated by the heater into the evaporation chamber.

13. The system of claim 1, further comprising a blower for blowing air heated by the heater into the reaction chamber.

14. The system of claim 1, wherein the system further comprises

A reservoir for storing the reaction solution and in fluid communication with the reaction vessel;

a pump for pumping the reaction solution from the reservoir to the reaction vessel; and

a valve for controlling flow of the reaction solution from the reservoir to the reaction vessel.

15. The system of claim 1, further comprising a valve between the reaction vessel and the purification vessel for controlling flow of a soluble component from the reaction mixture from the reaction vessel discharge to the purification vessel, the soluble component comprising the dissolved analyte.

16. The system of claim 15, wherein the valve comprises:

a first plate comprising an input port extending through the first plate and in fluid communication with a reaction vessel drain of the reaction vessel; and

a second plate positioned below the first plate, the second plate including a first output port extending through the second plate and in fluid communication with the purification vessel,

wherein in a first position of the first plate and the second plate, the input port of the first plate is aligned with the solid portion of the second plate, preventing soluble components from the reaction mixture from flowing from the reaction vessel discharge through the valve, and

wherein in the second position of the first plate and the second plate, the input port of the first plate is aligned with the first output port of the second plate, allowing soluble components from the reaction mixture to flow from the reaction vessel discharge through the valve to the purification vessel.

17. The system of claim 16, further comprising an actuator for moving the relative position of the first and second plates from the first position to the second position.

18. The system of claim 16, wherein the second plate further comprises a second output port extending through the second plate and in fluid communication with a drain reservoir,

wherein in a third position of the first plate and the second plate, the input port of the first plate is aligned with the second output port of the second plate, allowing soluble components from the reaction mixture to flow from the reaction vessel discharge through the valve to the discharge reservoir.

19. The system of claim 1, further comprising a nitrogen supply in fluid communication with the reaction vessel.

20. The system of claim 1, further comprising a nitrogen supply in fluid communication with the vaporization vessel.

Technical Field

The present invention relates to systems and methods for performing various chemical and physical manipulations that result in the automated extraction of analytes for quantitative measurement.

Background

Extraction (i.e., isolation) and quantification of certain nutrients in complex matrices, particularly in natural sources, can be challenging. In the early twentieth century, the government began to test foods by mandatory requirements of the clean food and drug act. In recent years, with the advent of the 1990 Nutrition Labeling and Education Act (NLEA), the food and drug administration required food manufacturers to provide nutritional information to consumers. Such nutritional information is provided in the form of nutritional labels on all packaged food products. Because of NLEA, food manufacturers need to analyze their products in order to provide accurate information about the nutritional composition to the customer.

The analysis of any food or feed product requires several initial or preliminary processes designed to chemically release, purify and concentrate the target analyte (select nutrients) from the physical and chemical matrix of the product. That is, before analytes can be identified and quantified by High Performance Liquid Chromatography (HPLC) or Gas Chromatography (GC), the hydrogen, ionic, and/or covalent bonds that bind the analyte to its physical and/or chemical matrix must be broken, and a sufficient amount must be collected.

Historically, these analytical procedures have been performed manually in an analytical laboratory by skilled laboratory technicians. More particularly, these processes have been directed to the quantitative extraction of analytes, such as Fat Soluble Vitamins (FSV), for final quantitation by spectrophotometry or more recently HPLC. FSV must be extracted in a non-polar solvent fraction free of water soluble compounds and most lipids. For example, the analysis of retinol (vitamin a) most often involves: (i) cleaving ester bonds by saponification, (ii) removing water-soluble compounds by biphasic separation and extracting the analyte, and (iii) concentrating the resulting analyte by evaporation of the solvent. The analysis becomes complicated by the need to perform each step without exposure to light of a selective wavelength and in the absence of oxygen.

The other major obstacle to analysis is the formation of an emulsion during biphasic separation. The emulsion effectively forms a third phase that is difficult to separate and prevents complete extraction of the analyte. Most emulsions will precipitate over time. If the emulsion is durable, additional steps, such as centrifugation or re-extraction, may be required to break the emulsion and completely extract the analyte. These are expensive analytical steps.

Another example of a need for several initial or preliminary processes to chemically release, purify, and concentrate a target analyte is total fat analysis. The method comprises the following steps: (i) hydrolysis in hydrochloric acid (HCl) solution, (ii) removal of water soluble compounds in biphasic separation of aqueous and organic solvent phases (in a Mojonnier flask), and (iii) evaporation of solvent for gravimetric quantification of separated fat. In other total fat processes, fat may be captured by lipophilic filters while allowing aqueous solutions to pass through. The residue and filter must be thoroughly dried before extraction with organic solvent. The drying step removes traces of water from the hydrolyzed sample and subsequently allows the nonpolar solvent to penetrate the otherwise polar hydrolyzed sample. After extraction, the solvent containing the fat was evaporated and the separated fat was quantified by weight. It will be appreciated that these methods are time consuming, financially burdensome and labor intensive.

In view of the difficulties and complexity of current methods associated with the extraction of analytes (e.g., FSV and total fat), there is a need for a self-contained, fully automated system and method for extracting analytes from complex samples.

Disclosure of Invention

Systems and methods for extracting an analyte from a sample are disclosed. In one embodiment, the system includes a reaction chamber including a reaction vessel having a column for receiving a sample and a reaction solution, a mixer for mixing the sample with the reaction solution, a filter, and a drain for passing soluble components (including dissolved analyte) from the reaction vessel.

In one embodiment, the purification chamber is located below the reaction chamber and includes a purification vessel having a column for receiving the dissolved analyte from the reaction vessel. A selective adsorbent is disposed in the purification vessel for retaining contaminants from soluble components of the reaction mixture and passing purified analytes therethrough.

An evaporation chamber is located below the purification chamber and includes an evaporation vessel for receiving purified analyte contained in a solvent from the purification vessel. The heater heats the evaporation chamber and evaporates the solvent from the purified analyte, which can then be transferred for quantitative measurement.

The above embodiments are merely exemplary. Other embodiments are within the scope of the disclosed subject matter.

Drawings

A more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments. For a further understanding of the nature and objects of the present invention, reference should therefore be made to the following detailed description, read in conjunction with the accompanying drawings, in which:

FIG. 1 depicts a perspective view of an exemplary automated system or extractor for extracting analytes for quantitative measurements;

FIG. 2 depicts a schematic cross-sectional view of the exemplary system shown in FIG. 1, including a reaction chamber, a shuttle valve transfer device, a purification chamber, and an evaporation chamber;

FIG. 3 depicts a perspective view of an exemplary reaction vessel, purification vessel, and evaporation vessel in parallel;

FIG. 4 depicts a schematic exploded view of exemplary components associated with a single station of a system including a reaction chamber, a shuttle valve transfer apparatus, a purification chamber, and an evaporation chamber;

FIG. 5 depicts an enlarged schematic view of a portion of an exemplary reaction vessel;

fig. 6 depicts a schematic exploded view of an exemplary purification vessel;

FIG. 7A depicts a cross-sectional view taken substantially along line 7A-7A, depicting the shuttle valve in a closed position;

FIG. 7B depicts a cross-sectional view taken substantially along line 7B-7B, depicting the shuttle valve in an open to vessel position; and

FIG. 7C is a cross-sectional view taken substantially along line 7C-7C, depicting the shuttle valve in an open to waste position.

Detailed Description

The present disclosure relates to an analyte extractor (or instrument) configured to automatically extract (i.e., separate) analytes from a complex matrix for subsequent quantitative analysis by, for example, chromatography, spectrophotometry, or gravimetry. Although the exemplary analyte extractor is primarily configured to extract (i.e., separate) fatty and lipid-soluble analytes, it should be recognized that the device is equally applicable to any device having as its primary function the release, extraction, purification, and separation of analytes that must be separated from a complex matrix. Further, while the exemplary analyte extractor includes various chambers/vessels/containers/processes in series for separating analytes for subsequent quantitative analysis, it will be appreciated that other embodiments may utilize fewer chambers/vessels/containers/processes to generate samples for further testing. For example, an analyte extractor may not utilize a vaporization chamber to produce analytes for subsequent analysis. Further, while the exemplary instrument includes up to four assay stations/locations a, b, c, d in parallel (i.e., in side-by-side relation) for performing an extraction process on four (4) complex samples, it will be appreciated that the analyte extractor may extract analyte from a sample using any number of assay stations or locations.

Fig. 1 depicts an exemplary analyte extractor 10 that includes a body or chassis 12 that allows for fixed mounting and removable mounting of various components of the extractor 10 in accordance with the teachings of the present disclosure. The exemplary analyte extractor 10 includes a plurality of vertically aligned chambers for mounting one or more similar components (i.e., components that perform the same or similar operations), including a reaction chamber 100, which is located above a purification chamber 200, which is located above an evaporation chamber 300.

A sample (e.g., a food or feed sample) may be deposited into and received by one or more reaction vessels 104a,104b,104c,104d in the reaction chamber 100. As used herein, the term "vessel" generally refers to, for example, a column, tube, or the like that can contain and allow passage of a fluid. As used herein, the terms column and tube are used interchangeably. A mixture of various solutions may be added to the sample in the reaction vessels 104a,104b,104c,104d, and then may be stirred (e.g., mixed) and heated to achieve a first function of dissolved sample analyte generation in the soluble components of the reaction mixture. The dissolved analyte flows continuously down through the filter to one or more removable purification vessels (e.g., columns, tubes, etc.) 204a,204b,204c,204d associated with the purification chamber 200, which are vertically aligned along the same axis and below the respective reaction vessels 104a,104b,104c,104d, such that a second operation can be performed to produce a purified analyte contained in a solvent. Similarly, purified analyte contained in a solvent in the second row or purification chamber 200 can flow continuously down one or more containers (e.g., flasks) 304a,304b,304c,304d in yet another row associated with the evaporation chamber 300, which are vertically aligned along the same axis and below the respective purification vessels 204a,204b,204c,204d, so that yet another operation can be performed.

In the described embodiment, the analyte extractor 10 may include a plurality of columns/stations/channels a, b, c, d for integrating multiple vessels (e.g., columns, tubes, etc.) and containers (e.g., flasks) in parallel. Thus, a plurality of samples corresponding to the number of stations can be processed simultaneously, thereby greatly improving throughput. Control inputs to the analyte extractor 10 may be made via a display, command, input screen, or touch screen 22. All variables associated with a method or process may be entered through the display/screen 22. Each of these system components and method steps will be discussed in greater detail in subsequent paragraphs.

Fig. 2, 3 and 4 depict detailed schematic views of an exemplary analyte extractor 10 of the present disclosure depicting relevant internal details and components of the instrument. More particularly, a pump P may be associated with the valve V and the flow meter M to inject a volume of liquid/solution into the reaction vessels 104a,104b,104c,104d of the reaction chamber 100. In one embodiment, diffusion nozzles may be located at the inlets of reaction vessels 104a,104b,104c,104d to bias the pumped solution toward the inner walls of the reaction vessels to flush and dissolve analyte material disposed along the inner walls of the reaction vessels.

The heater H is operable to heat the sample/mixture within one or more chambers 100,300, effectively forming an oven in each chamber 100, 300. The blower B is operable to circulate air within one or more of the chambers 100, 300. One or more temperature sensors T1 and T2 control the temperature in the chamber 100, 300. Temperature sensor S_TMay also be provided in, on, or integrated with the reaction vessels 104a,104b,104c,104d to provide temperature feedback in proximity to or within the processed/evaluated sample analyte.

One or more actuators a may be used to open/close the set of shuttle valves 150 at appropriate intervals during analyte extraction. Multiple reservoirs R may be employed_L1,R_L2,R_L3,R_L4And a corresponding valve V to mix the various solutions with the sample at least inside the reaction chamber 100. Furthermore, although in the exemplary embodiment, reservoir R_L1,R_L2,R_L3,R_L4Each of which includes a valve V, it will be appreciated that a single valve V may be used to control two or more reservoirs R_L1,R_L2,R_L3,R_L4The associated flow. A processor 20, such as a microprocessor, is operable to control all processes within the system. Similarly, while a single processor 20 is shown as controlling the operations associated with each chamber 100,200,300, it will be appreciated that several microprocessors may be employed to control the independent functions of the analyte extractor 10. Finally, a power source (not shown) can be used to activate the pump P, heater H, blower B, valve V, actuator A, temperature sensor S_TT1 and T2, pressure sensor S_PLiquid sensor S_LA set of shuttle valves 150, and a processor 20 of the analyte extractor 10. Similarly, the pump P, valve V and flow meter M are operatively coupled to the processor 20 so as to be contained in the fluid reservoir R_L1,R_L2,R_L3,R_L4The exact flow and amount of solution in (a) may be supplied to the reaction vessels 104a,104b,104c,104 d.

Reaction chamber

The analyte extractor 10 performs a number of critical operations to separate target analytes from complex samples. In a first process, i.e., in reaction chamber 100, the sample may be exposed to one or more solutions while being stirred and heated in an oxygen-free environment. This step releases the target analyte from the complex matrix. Fat soluble vitamin analysis typically releases vitamins through an ethanol saponification reaction, while total fat analysis typically releases fat through an HCl hydrolysis reaction. The analytes produced by these reactions can be dissolved in complex solution mixtures that bind insoluble residues. The mixture may be filtered before it is transferred to a purification vessel (the analyte extractor or the second chamber 200 of the instrument 10).

The reaction vessel 104 has a unique design that allows the separation of the liquid portion from the insoluble residue. For ease of discussion, a single reaction vessel 104a will be described in detail based on the consensus that adjacent reaction vessels 104b,104c,104d may be substantially identical and that no additional description is required or warranted. Thus, when referring to one reaction vessel in the reaction chamber 100 and/or one component associated with the purification chamber 200 and the evaporation chamber 300, it is understood that the described vessels or steps may be applied to all of the same components associated with adjacent assay stations. The reaction vessel 104a may be removable such that it may be placed on a balance/scale and the sample may be weighed directly in the vessel 104 a.

Reaction vessel 104a may be chemically inert and designed to withstand strong acids, bases, and organic solvents over a temperature range of, for example, between 20 ℃ and 105 ℃. Prior to reaction, the reaction vessel 104a may be closed (e.g., automatically closed) by capping a chemically inert reactor vessel lid (i.e., lid, plug, top, etc.) 108a and closing the top opening of the vessel 104 a. Each reactor vessel lid 108a may contain a temperature sensor S_TAnd a pressure sensor S_PAnd orifices (i.e., ports, orifices, apertures, etc.) 112a for the exhaust line and the liquid supply line. The reaction vessel 104a may be completely sealed and, with feedback from each of these sensors, provide complete control of the internal environment. To protect sensitive analytes such as vitamin A and vitamin E, inert gases such as nitrogen (N) may be introduced₂) An oxygen-free environment is created in the reaction vessel.

Each reaction vessel 104a may be removable to facilitate removal, cleaning, sterilization, and loading of sample material (not shown). The reaction vessel 104a may include a cylindrical reaction vessel column 106a made of, for example, borosilicate glass, a reactor vessel lid 108a having a plurality of apertures 112a, and a removable reaction vessel base 110 a. It will be understood that the scope of the present invention includes reaction vessels 104a having a shape other than a cylindrical column. Except for receiving the temperature sensor S_TAnd a pressure sensor S_PIn addition, the orifice 112a may receive fluid or exhaust gas/volatiles from the sample mixture of the reaction vessel 104 a. The liquid sensor S may be provided through the top or bottom of each vessel or container_LTo ensure that the liquid has drained or evaporated from the respective vessel or container. For example, in FIG. 4, reaction vessel 104aMay include a liquid sensor S passing through the removable reaction vessel base 110a_LTo indicate when fluid has been drained from reaction vessel 104 a. Alternatively or additionally, a liquid sensor S_LMay be disposed in a flexible line between reaction vessel drain (e.g., drain) 116a and shuttle valve 150. When the refractive index is changed, the liquid sensor S_LIt can be determined that liquid is no longer present in the tube and therefore no longer remains in reaction vessel 104 a.

While the exemplary borosilicate glass reactor column 106a may be configured for cleaning, sterilization and reuse, it will be appreciated that other disposable materials may be employed. For example, a clear polypropylene cylindrical column with built-in components may be employed to facilitate rapid deployment and reuse of the reaction vessel 104 a. That is, the disposable reaction vessel 104a may include a housing having a receiving chamber for receiving the temperature sensor S_TPressure sensor S_PAnd one or more fluid fill line ports 112a, in the reactor vessel closure 108 a. The disposable vessel 104a may also include a reaction vessel base 110a, the reaction vessel base 110a including a mixer driver 132a, a mixing agitator bar 130a, a requisite reaction vessel filter 140, and a reaction vessel drain 116 a. An O-ring seal may or may not be required, as other sealing methods may be employed and reaction vessel base 110a may or may not be removable.

In the depicted embodiment, the reactor vessel lid 108a may be fixed to the instrument and may be capable of applying a downward force to seal the reactor vessel lid 108a, the reaction vessel column 106a, and the removable reaction vessel base 110a of the reaction vessel 104 a. The reactor vessel lid 108a comprises at least two apertures 112a arranged to communicate with the fluid reservoir R_L1,R_L2,R_L3,R_L4And at least one nitrogen gas (N)₂) The supply portions are in fluid communication. Nitrogen gas N can be introduced₂Into the reaction vessel 104a to create an oxygen-free or oxygen-depleted environment. Thus, evaporation of the solvent does not form a combustible gas, as the ratio of solvent to oxygen can be kept below the combustion level. From nitrogen (N)₂) Can also serve to facilitate flow of dissolved analyte through reaction vessel filter 140a and reaction vessel drain 116a of the particulate reaction vesselThe application is as follows.

The capsule lid 108a may be made of plastic. In one embodiment, the plastic may be a polytetrafluoroethylene polymer, commonly referred to as Teflon @ (Teflon @, a registered trademark of E.I. du Pont de Nemours and Company, Wilmington, Del.). Since the vessel lid 108a is exposed to large temperature variations, i.e., between room temperature and over one hundred degrees celsius (100 ℃), the effects of thermal expansion must be considered to ensure a gas-tight and liquid-tight seal with the reaction vessel column 106 a. With respect to the reactor vessel lid 108a, an elastomeric or rubber O-ring may seal between the wall surface of the reactor vessel column 106a and the reactor vessel lid 108 a. This arrangement and geometry may improve seal integrity as the sealing interface may be compressed due to thermal growth or differences in the coefficient of thermal expansion between the plastic vessel lid 108a and the borosilicate glass reaction vessel column 106 a.

Fig. 5 shows a detailed schematic view of the reaction vessel base 110 a. The reaction vessel base 110a may also be made of polytetrafluoroethylene polymer or a plastic material (e.g., teflon @). O-ring seal 118a may be disposed between an outer peripheral surface 122a of reaction vessel column 106a and an inner wall of reaction vessel base 110 a. Since the plastic reaction vessel base 110a may grow larger than the borosilicate glass reaction vessel column 106a, an outer ring or sleeve of reinforcing material 114a may surround the plastic reaction vessel base 110a to limit its outward growth. The material selected for the outer ring or sleeve 114a should have a lower coefficient of thermal expansion than the plastic reaction vessel base 110, and preferably have a similar rate of thermal expansion as borosilicate glass. In the depicted embodiment, the outer ring or sleeve 114a may be made of a metal such as stainless steel. Thus, the seal integrity may be maintained or improved by the inwardly directed radial force 124a exerted by the metal outer ring 114 a. To further improve seal integrity, the O-ring seal 118a is received within a groove 120a having a substantially dovetail-shaped cross-sectional geometry. This configuration captures the O-ring seal 118a when the reaction vessel column 106a is slid into and received in the cavity of the plastic reaction vessel base 110 a. That is, the efficacy of the dovetail groove 120a to maintain a sealing interface along the outer peripheral surface 122a of the reaction vessel column 106a, particularly during assembly of the plastic reaction vessel base 110 a.

Reaction vessel 104a may include a mixing stir bar 130a and a concentric mixer driver 132a, which may be mounted below reaction vessel base 110a and operable to mix fluid with a sample in reaction vessel 104 a. More particularly, the mixing system includes a coaxially aligned mixing agitator bar 130a and a mixer driver 132 a. Reaction vessel discharge 116a of reaction vessel base 110a may be aligned with and in fluid communication with extraction port 117a formed within the housing of mixer driver 132 a. Further, an O-ring 134a may be disposed at the interface between reaction vessel discharge 116a of reaction vessel 104a and extraction port 117a of mixer drive 132a to provide a fluid seal during operation. This design allows the effluent to exit at the bottom of the reaction vessel base 110 a.

A high torque mixing system is preferred because of the significant viscosity increasing reactions that occur when combining certain samples and chemical solutions. In addition, the mixing system must be robust enough to completely mix the biphasic solution in a vessel of limited diameter. The mixer motor has the ability to change speed and direction, thus allowing the magnet to break loose and rotate under high viscosity conditions. The mixing agitator bar 130a may be magnetic, i.e., have north and south poles that repel or attract relative to the poles generated by the magnetic mixer driver 132 a. More particularly, the mixer driver 132a may define an annular electrical winding that surrounds the extraction port 117a and generates an alternating magnetic flux field for driving the mixing stirring rod 130a about the axis of rotation. As one or more saponification fluids or solvents are added to the reaction vessel 104a, the mixing stirring rod 130a agitates the sample.

In the depicted embodiment, the mixer driver 132a portion of the mixers 130a,132a may be disposed below the reaction vessel 104a and outside the reaction chamber 100, which may form an oven when heated. As a result, the mixer driver 132a is not affected by the heat of the reaction chamber 100. In addition, since a solvent such as ethanol or hexane is used in the reaction chamber 100, the current-driven magnetic mixer driver 132 cannot generate an electric spark in the reaction chamber 100 that may contain a combustible gas.

Reaction vessel 104a may be broken down into reaction vessel column 106a and reaction vessel base 110a, which allows reaction vessel filter 140a of the reaction vessel to be placed between them. The compressive force exerted by the reactor vessel lid 108a seals and secures the periphery of the reaction vessel filter 140a of the reaction vessel so that particles cannot bypass the reaction vessel filter 140a of the reaction vessel. The reaction vessel filter 140a of the selective reaction vessel is capable of filtering insoluble particulate matter from dissolved analyte material. More particularly, when performing vitamin analysis, the reaction vessel filter 140a of the reaction vessel separates the liquid from the solid. Further, when performing fat analysis, the reaction vessel filter 140a of the reaction vessel quantitatively retains the lipid fraction while removing the unnecessary aqueous fraction to be discarded.

When the reaction vessel 104a is removed from the reaction chamber 100, filled with sample material and weighed, the reaction vessel filter 140a of the reaction vessel may be used as a temporary valve. That is, since the reaction vessel 104a must contain a dry or wet sample when loaded, weighed and subsequently reassembled into the reaction chamber 100, the reaction vessel filter 140a of the reaction vessel prevents the sample or a portion of the sample from escaping through the reaction vessel discharge 116 a. The composition of reaction vessel filter 140a may vary depending on the chemical resistance characteristics and the type of analysis being performed.

For example, when performing a fat analysis on a food sample, reaction vessel filter 140a may be made of a filter medium having the ability to retain particles of two microns (2 μm) and larger. Typically, when such an analysis is performed, reaction vessel filter 140a will range between about two microns (2 μm) to about fifteen microns (15 μm). When performing vitamin analysis, the filter media of reaction vessel filter 140a may be made of filter material having a pore size of less than about eight microns (8 μm). Typically, the filter media of reaction vessel filter 140a ranges between about eight microns (8 μm) to about thirty microns (30 μm). The retention of the particles when performing vitamin analysis need not be comprehensive. Although it is important that the fines do not clog the fines lines and valves, it is not important that all of the fines remain in the reaction vessel filter 140a, as the entire saponified mixture of liquid and ultra-fine particles is transferred to the purification vessel 204a, as opposed to fat analysis. The purification vessel 204a not only retains polar compounds, but also filters out any fine particles that pass through the reaction vessel filter 140 a.

Shuttle valve

It will be appreciated that the sample and dissolved analyte remain in the reaction chamber 104a for a prescribed time (e.g., residence time) while the reaction occurs in the reaction vessel 104 a. In one embodiment, a timer is provided to determine a dwell time associated with operation of, for example, the mixer, pump and heat source, and to provide a dwell signal indicative of the operating time of each.

In the depicted embodiment, this may be accomplished by a shuttle valve 150 that prevents gravity flow of dissolved analyte from reaction vessel 104a for a specified residence time. Fig. 7A-7C depict schematic cross-sectional views of each of the ports 156a-156d,158a-158d along the length of the shuttle valve 150 and through the shuttle valve in different configurations of the shuttle valve 150. Arrows 7A-7A,7B-7B,7C-7C show the direction of the cross-section and do not provide information about the kinematics of the shuttle valve operation. It will be appreciated from a review of the subsequent paragraphs that the plates 152,154 of the shuttle valve 150 slide orthogonally relative to the direction of the arrows 7A-7A,7B-7B, 7C-7C.

In fig. 3 and 7A-7C, the shuttle valve 150 may include a pair of slide plates, an upper or first plate 152, and a lower or second plate 154, wherein the first plate 152 includes ports 156a,156b,156C,156d spaced horizontally in the plane of the plate 152. The plates 152,154 are interposed between the reaction vessel discharge 116a,116b,116c,116d of the respective reaction vessel 104a,104b,104c,104d and the input port 202a,202b,202c,202d of the respective purification vessel 204a,204b,204c,204 d.

Inspection of the configuration of the plates 152,154 shown in fig. 7A (closed position) reveals that the ports 156a,156b,156c,156d of the first plate 152 are closed-end or against the upper surface of the second plate 154. Thus, when shuttle valve 150 is in the closed position, dissolved analyte is prevented from flowing from reaction vessel discharge 116a,116b,116c,116d of respective reaction vessels 104a,104b,104c,104d to input ports 202a,202b,202c,202d of respective purification vessels 204a,204b,204c,204 d.

Inspection of the configuration of plates 152,154 shown in fig. 7B reveals that shuttle valve 150, when in the open to waste position, facilitates passage of fluid through first plate 152 and second plate 154 by way of aligned pairs of ports 156a,157a,156B,157B,156c,157c,156d,157d in plates 152,154, respectively. That is, actuator A moves the relative positions of the plates 152,154 such that ports 156a,156b,156c,156d in one plate 152 are aligned with ports 157a,157b,157c,157d of the opposite plate 154. It will be appreciated that the input ports 156a,156b,156c,156d align with the output ports 157a,157b,157c,157d located on the lower plate 154 to allow fluid to flow from the reaction vessels 104a,104b,104c,104d through the plates 152,154 towards the drain reservoirs. This may be accomplished by moving the second or lower plate 154 in one direction, such as to the left in the direction of arrow L (fig. 7B), while maintaining the position of the upper plate 152, i.e., remaining stationary. In another embodiment, the second or lower plate 154 may remain stationary while the first or upper plate 152 moves to the right. A potential use of the discharge to a waste location is to remove solvent vapors from the reaction vessel or to remove unwanted liquids from the reaction vessel.

Inspection of the configuration of plates 152,154 shown in fig. 7C reveals that shuttle valve 150 is in the open to vessel (purge) position, which facilitates passage of fluid through first plate 152 and second plate 154 by way of aligned pairs of ports 156a,158a,156b,158b,156C,158C,156d,158d in plates 152 and 154, respectively. That is, actuator A moves the relative positions of the plates 152,154 such that the ports 156a,156b,156c,156d in one plate 152 are aligned with the output ports 158a,158b,158c,158d of the opposite plate 154. It will be appreciated that the input ports 156a,156b,156c,156d align with output ports 158a,158b,158c,158d located on the lower plate 154 to allow dissolved analyte to flow from the reaction vessels 104a,104b,104c,104d through the plates 152,154 to the respective purification vessels 204a,204b,204c,204 d. This may be accomplished by moving the second or lower plate 154 in one direction, e.g., to the right in the direction of arrow R (fig. 7C), while maintaining the position of the upper plate 152, i.e., stationary. In another embodiment, the second or lower plate 154 may remain stationary while the first or upper plate 152 moves to the left. Thus, the shuttle valves simultaneously control the flow between the reaction vessels 104a,104b,104c,104d and the purification vessels 204a,204b,204c,204 d.

The low profile geometry of the shuttle valve 150 allows the valve 150 to be installed below the reaction chamber 100 while remaining in close proximity to the reaction vessel discharge 116a,116b,116c,116d of the reaction vessels 104a,104b,104c,104 d. Furthermore, connecting the reaction vessel discharge 116a,116b,116c,116d to the purification vessels 204a,204b,204c,204d using small inner diameter tubing ensures a minimum air gap between them. This ensures that liquid does not migrate to the valve area when the reaction chamber 100 is in operation. Finally, the shuttle valve 150 may be pneumatically actuated to reduce the possibility of electrical sparking in areas that may contain vaporized solvent and potentially flammable/combustible gases.

Purification chamber

In purification chamber 200, analyte extractor 10 separates a desired portion of the dissolved analyte from reaction vessel 104a from an undesired portion of the dissolved analyte from reaction vessel 104 by passing the dissolved analyte through purification vessel 204 filled with selective adsorbent 216a (e.g., a solid phase filter material such as siliceous earth, also commonly referred to as Diatomaceous Earth (DE), or other chromatographic medium or alumina). Prior to passing the dissolved analyte through the purification vessel 204, the selective adsorbent can be adjusted to retain the polar compound in the purification vessel 204 while allowing more of the non-polar target analyte to pass through the purification vessel 204. This can be accomplished by passing specific amounts of water and ethanol through the selective adsorbent prior to or concurrently with the sample.

As shown in fig. 1, 3 and 6, the purification chamber 200 defines a cavity for mounting four (4) purification vessels 204a,204b,204c,204d, which may be arranged substantially horizontally across the purification chamber 200. Continuing with the above description, a single purification vessel 204a will be described based on the consensus that adjacent vessels 204b,204c,204d may be substantially identical and that no additional description is required or warranted. Thus, when referring to one purification vessel 204a in a purification chamber 200, it is understood that the described vessels apply to all vessels in adjacent assay stations.

The purification vessel 204a may be configured to receive dissolved analyte from the reaction vessel 104a after analyte reaction and filtration in the reaction chamber 100. More particularly, purification vessel 204a may be in fluid communication with reaction vessel discharge 116a of reaction vessel 104a through shuttle valve 150.

The purification vessel 204a includes a polymer (e.g., polypropylene) cylindrical purification vessel column 208a having a purification vessel discharge (e.g., conical nozzle) 210a at one end and a top opening 212a at the other end, the top opening 212a being dimensionally equivalent to the diameter of the purification vessel column 208 a. It will be understood that the scope of the present invention includes a purification vessel 204a having a shape other than a cylindrical column. Although in the disclosed embodiment, the purification vessel 204a can be a polymer, it is to be understood that the purification vessel 204a can be any flexible or rigid disposable container.

The purification vessel column 208a may be configured to receive: (i) a lower purification vessel filter 214a for placement above the purification vessel drain 210a, (ii) a volume of selective adsorbent (e.g., Siliceous Earth (SE))216a, (iii) a purification vessel diffuser 218a, and (iv) a purification vessel lid (i.e., lid, plug, top, etc.) 220a for controlling the flow of dissolved analyte and nitrogen into the purification vessel column 208 a. The lower purification vessel filter 214a and purification vessel diffuser 218a are used to hold the selective adsorbent 216a without allowing any filter material to pass through the purification vessel filter 214a or the purification vessel diffuser 218 a. The pores of the lower purification vessel filter 214a and the purification vessel diffuser 218a must be small enough to hold the selective adsorbent material.

Dissolved analyte from reaction vessel 104a enters purification vessel column 208a via reactor vessel lid orifice 224a in purification vessel lid 220 a. In one embodiment and as shown in fig. 3, the input port 202a can feed the mixture from the reaction vessel discharge 116a to the purification vessel closure 220a of the purification vessel column 208 a. Once through the reactor vessel lid orifice 224a, the purification vessel diffuser 218a diffuses or spreads the analyte solution to prevent the formation of a flow path through the selective adsorbent 216a (similar to the erosion caused by flowing water). Thus, the analyte material is spread on top of selective adsorbent 216a in a substantially uniform manner. Once the dissolved analyte from reaction vessel 104a passes through purification vessel 204a, the purified analyte contained in the solvent flows sequentially through purification vessel drain 210a to one or more containers (e.g., flasks) 304a,304b,304c,304d in evaporation chamber 300.

Evaporation chamber for evaporating liquid from purified analyte material

In evaporation chamber 300, the solvent can be evaporated from the purified analyte, such that the purified analyte can be collected for subsequent quantification (e.g., by HPLC or GC). In fig. 4, an evaporation vessel (e.g., flask) 304a is in fluid communication with and receives purified analyte material from purification vessel 204a (i.e., from purification vessel drain 210 a). It will be understood that the scope of the present invention includes vaporization vessel 304a having a shape other than a flask. The purified analyte mixture comprises a solvent that evaporates in an oxygen-free environment within evaporation vessel 304. That is, the vaporization container 304a may be filled with an inert gas (e.g., nitrogen N)₂) Via nozzle 308 a. The nozzle 308a may be provided in combination with a cap (not shown) that is inserted into the opening of the vaporization container 304a, with a vent port in the cap (not shown) allowing inert nitrogen (N)₂) The high velocity flow of (a) moves the solvent within the container 304a and promotes evaporation.

In addition to the solvent moving within vaporization container 304a, vaporization container 304a may be heated to increase the rate of vaporization. The container 304a may be continuously purged with nitrogen to protect the analyte from oxidation. To protect the light sensitive analytes from ultraviolet light of selected wavelengths, an ultraviolet-protected polycarbonate door may cover the chambers 100 and 300.

In fig. 2 and 4, the conduits from the heater H may be bifurcated so that the flow of heated air may be directed to the reaction vessel 104a in the reaction chamber 100 and the evaporation vessel 304a in the evaporation chamber 300. Temperature sensors T1, T2, S located in the reaction chamber 100 and the evaporation chamber 300_TThe temperature signal is provided to the processor 20. These signals represent the instantaneous temperature within each chamber 100,300 and within each reaction vessel 104a,104b,104c,104d and each vaporization container 304a,304b,304c, and 304 d. The processor 20 may compare these signals to predetermined temperature values stored in the processor memory. The processor 20 evaluates the difference or error signal between the stored temperature values and the actual/instantaneous temperatures to increase or decrease the temperature in the respective chamber 100,300 and/or reaction vessel 104a,104b,104c,104d and vaporization vessel 304a,304b,304c,304 d.

Temperature sensors Tl, T2 and S_TCan be located in the analyte extractor 10 for the purpose of plotting the temperatures in the reaction chamber 100 and the evaporation chamber 300. With respect to the reaction chamber 100, the depicted embodiment shows a temperature sensor T1 to determine the temperature within the chamber 100, while a temperature sensor S_TThe temperature in each upper end cap is measured to obtain a temperature reading from within each reaction vessel 104a,104b,104c,104 d. While these locations provide reasonably accurate images of the temperature within the reaction chamber 100 and within the vessels 104a,104b,104c,104d, it will be appreciated that other locations may provide more direct or more accurate temperature measurements.

For example, in an alternative embodiment, a thermocouple may be attached to the reaction vessel column 106a of each of the vessels 104a,104b,104c,104d such that the temperature of the sample mixture may be measured within the respective vessel 104a,104b,104c,104 d. Of course, this may assume that borosilicate glass has a sufficiently low R (resistivity) value and does not function as an insulator. In yet another embodiment, a temperature sensor or thermocouple may be integrated within the plastic reaction vessel base 110a of each vessel 104a,104b,104c,104d such that the temperature may be measured at the bottom of the respective vessel 104a,104b,104c,104 d.

Fluid reservoir R_L1,R_L2,R_L3,R_L4One or more strongly basic or acidic fluids may be included, such as potassium hydroxide (KOH) or hydrochloric acid (HCl). Alternatively, the reservoir R_L1,R_L2,R_L3,R_L4May contain one or more solvents, including water (H)₂O), ethanol (CH)₃CH₂OH) and Hexane (CH)₃(CH₂)₄CH₃). From reservoir R_L1,R_L2,R_L3,R_L4And/or the flow from the nitrogen source may be provided or activated by an external pump P and/or controlled by one or more valves V and/or flow meters M. In addition to the orifice 112a for containing a flow of fluid or gas, the reactor vessel lid 108a may include a port for receiving a pressure sensor S_PAt least one orifice.

The processor or controller 20 may be responsive to the temperature sensor signal S provided by each of these sensors_TPressure sensor signal S_PAnd a liquid sensor signal S_LTo change the reaction chamber100. Temperature, pressure and flow rate within the purification chamber 200 and the evaporation chamber 300. With respect to the temperature in the reaction chamber 100, an alternative or second temperature sensor T1 (see fig. 2) may be disposed in the reaction chamber 100 rather than through the reactor vessel lid 108a of the reaction vessel 104 a. The temperature in the reaction chamber 100 can be changed by controlling the outputs of the heater H and the blower B. Accordingly, the processor 20 may be responsive to temperature signals from one or more temperature sensors.

The temperature sensor Tl may vary or vary the output of the heat source H with the flow of the blower B. In one embodiment, a heat exchanger may be connected to the heater H and a blower may direct air through the heat exchanger to produce heated air. It will be appreciated that the flow of heating air may be bifurcated such that some or all of the heating flow H is conducted or caused to react_SCan be directed to the reaction chamber 100 and some or all of the flow H during evaporation of the liquid contained in the sample_EMay be directed to the evaporation chamber 300. Thus, processor 20 can direct flow from heater H to either of chambers 100,300 via bifurcated conduit (BD). Processor 20 may be responsive to the pressure signal to increase nitrogen (N) during the reaction₂) To improve or increase the flow rate through reaction vessel discharge 116 a. This may also be used to introduce nitrogen (N) after the reaction in the reaction chamber 100₂) And injected into the subsequent purification chamber 200.

Designing automation systems and methods is a complex task involving a series of inventive steps that are not apparent at the beginning of a project. Developing an automatically executing instrument overcomes many significant difficulties and challenges.

The reaction mixture resulting from the reaction necessary to chemically release the analyte is not always compatible with the next process. For example, the requirements for conditioning SPE (solid phase extraction) columns are generally incompatible with reaction mixtures designed for chemical release of analytes. The passage of the analyte from the reaction chamber with both a solid portion and a liquid portion is not compatible with the valve function necessary to transfer the analyte to the next step. Previous solutions have been to perform the reaction before or on filtration so that the liquid will only pass under certain conditions. Later it was found that changes in the reaction and specialized filtration design were required. More particularly, variations in unique filter designs, special hybrid configurations, and solutions are required.

For example, the solution passing through the filter comprises a complex mixture of analytes and contaminants in aqueous and organic solvent solutions. To separate the analytes, SPE columns were used. The stationary phase of the SPE is capable of retaining contaminants, water, and other polar solvents while allowing a non-polar solvent (e.g., hexane) to elute the analyte for transfer to the evaporation chamber. The resulting system includes a reaction vessel having a bottom portion configured to be removably released to facilitate filter removal/replacement and sample introduction. In addition, the development of valve systems facilitates the transfer of solutions to the next chamber or disposal. Finally, an SPE column was used for purification, which communicates with the flask in the evaporation chamber and the solvent was removed by nitrogen in combination with a directed heat input.

Examples of analyte extraction

Vitamin preparation

The analyte extractor 10 of the present disclosure is capable of extracting analytes from complex matrices. One example is the extraction of vitamins a and E from infant formula. The infant formula may be reconstituted with water and the sub-sample (aliquot) and combination of antioxidants then weighed in reaction vessels 104a,104b,104c and 104 d. Each of the reaction vessels 104a,104b,104c and 104d may then be assembled into the reaction chamber 100. Once the reaction vessels 104a,104b,104c,104d are placed on the instrument 10, the respective reactor vessel lids 108a,108b,108c,108d may be combined with the respective reaction vessels 104a,104b,104c,104d (i.e., a downward force applied via the mounting brackets, effecting a fluid-tight seal with each of the reaction vessels 104a,104b,104c,104d,104 d). The following process is automatically controlled by the processor 20: the saponification solution (KOH and ethanol) is added, mixed and heated to 75 ℃ for 30 minutes, water is added, cooled to 60 ℃, the reaction mixture is passed through a filter and the liquid is allowed to pass into a SPE column containing celite, vitamins a and E (leaving contaminants) are eluted from the SPE column with hexane, and the vitamins are passed into a round bottom flask of an evaporation chamber 300, then the solvent is evaporated by a strong nitrogen flow and a heat source concentrated at the bottom of the flask. The separated oil containing vitamins a and E was manually re-dissolved in hexane and injected into HPLC for quantification.

Total fat analysis

Another example is total fat analysis by acid hydrolysis. The analyte extractor 10 recovers the total fat by combining the digestion (HCl) and extraction processes in the reaction chamber 100 with the separation capacity of the SPE. Ethanol can be used to displace residual water and to fill polar gaps with n-hexane, allowing n-hexane to penetrate the sample residue and filter, ultimately dissolving fat. Continuous stirring in combination with heated solvent greatly improves the extraction capacity. The SPE column bound the water/ethanol solvent and components and allowed hexane to elute with the fat. This selective capability of the SPEs allows the analyte extractor 10 to bypass the traditional drying step of hydrolyzed samples, allowing for total fat analysis to be performed in a single device/instrument.

The steps involved in analyzing the total fat begin with the breakdown of the sample in the reaction vessel 104 a. An optional multi-layer reaction vessel filter 140a may be placed in reaction vessel 104a above reaction vessel discharge 116a prior to adding the sample. The reaction vessel filter 140a of the reaction vessel comprises a combination of rigid and flexible layers that provide structural and Loft benefits. The Loft prevents the sample from clogging the filter during filtration, while the rigidity of the reaction vessel filter 140a of the reaction vessel prevents it from moving under the reaction vessel column 106 a. The sample can then be added and reassembled into the mounting bracket within the reaction chamber 100. In the total fat analysis, hydrochloric acid (HCl) may be contained in reservoir R_L1,R_L2,R_L3,R_L4And may be automatically added to the reaction vessel 104 a.

The sample may be heated in HCl solution to release bound fat, enhanced by continuous mixing. Once the process is complete, the aqueous solution may be filtered for disposal through shuttle valve 150. By mixing and heating the sample in the reaction chamber 100, the chemical breakdown of the sample can be optimized and the fat is completely released from the sample matrix. Chemical decomposition also reduces the formation of gel-like materials that may clog the filter. Because the chemical bonds are broken and contaminants are removed, the analyte extractor 10 may filter large samples through a relatively small filter that only allows aqueous solutions to pass through.

The analyte extractor 10 bypasses the drying step by integration of the SPE cartridge. Rather than drying the sample as discussed in the background section of this disclosure, analyte extractor 10 automatically adds solvents (e.g., ethanol and hexane) to the sample residue held on reaction vessel filter 140a of the reaction vessel. The hydrophilic nature of ethanol combines with water to form a new solvent that is compatible with hexane, enabling hexane to extract fats. After extraction of the wet residue with a solvent, the extracted solvent comprises a dissolved mixture of moderately polar substances and fat.

Therefore, fat must be separated from the non-fat components. In the present disclosure, this step may be performed by SPE column, where polar and moderately polar contaminants are separated from non-polar fatty components. In addition, the SPE column interacts with the mixed sample solution, allowing only the non-polar solvent containing the fat to leave the SPE column and enter the evaporation flask. Once the solvent and fat entered the evaporation flask, the solvent evaporated, leaving only the fat for further analysis.

The analyte extractor 10 is unique compared to other total fat analysis methods. For example, the analyte extractor 10 may perform total fat analysis with a single instrument that does not require a separate drying step. This distinguishes analyte extractor 10 from other extraction methods that require a combination of at least two instruments and an oven drying step to complete the total fat analysis.

It should be understood that various changes and modifications to the embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present disclosure and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

While several embodiments of the present disclosure have been disclosed in the foregoing specification, it will be appreciated by those skilled in the art that many modifications and other embodiments of the disclosure to which the disclosure pertains will come to mind, having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed above and that modifications and other embodiments are intended to be included within the scope of the appended claims. Moreover, although specific terms are employed herein, as well as in the claims that follow, they are used in a generic and descriptive sense only and not for purposes of limiting the disclosure, nor the claims that follow.