Systems and methods for extracting analytes from a sample

Systems and methods for extracting an analyte from a sample. The system includes a reaction vessel for receiving the sample and a reaction solution, a mixer for mixing the sample with the reaction solution, a filter and a drain for passing soluble components from the reaction mixture, including the dissolved analyte, from the reaction vessel. A purification vessel is located below the reaction vessel. A selective sorbent is disposed in the purification vessel for retaining contaminants from the soluble components from the reaction mixture and passing a purified analyte. An evaporation container is located below the purification vessel. A heater heats the evaporation chamber and evaporates the solvents from the purified analyte, which can then be quantitatively measured.

TECHNICAL FIELD

The present invention is directed to systems and methods that perform various chemical and physical operations resulting in the automated extraction of an analyte in preparation for quantitative measurement.

BACKGROUND

The extraction (i.e., isolation) and quantitation of certain nutrients from complex matrices can be challenging, particularly from natural sources. In the early 1900s the government started mandating testing of foods through the Pure Food and Drug Act. More recently, with the advent of the 1990 Nutrient Labeling and Education Act (NLEA), the Food and Drug Administration has required food manufacturers to provide nutrient information to consumers. Such nutrient information is provided in the form of a nutrition label on all packaged foods. As a consequence of the NLEA, food manufacturers are required to analyze their products so that accurate information about the nutrient content can be provided to customers.

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

Historically, these analytical processes have been performed manually in an analytical laboratory by skilled laboratory technicians. More specifically, these processes have been performed to quantitatively extract analytes such as Fat Soluble Vitamins (FSVs), leading to final quantitation by either spectrophotometry or more recently HPLC. FSVs must be extracted in a non-polar solvent fraction that is free from water soluble compounds and most lipids. For example, the analysis of Retinol (Vitamin A) most commonly involves: (i) the cleavage of ester linkages through saponification, (ii) removal of water-soluble compounds and extraction of the analyte by bi-phase separation, and (iii) concentration of the resultant analyte by evaporation of the solvent. The analysis is complicated by a requirement to conduct each step without exposure to selective wavelengths of light and in the absence of oxygen.

Other major impediments to the analysis are formation of emulsions during bi-phase separations. Emulsions effectively forms a third phase that is hard to separate and which prohibits complete extraction of the analyte. Most emulsions will settle over time. If emulsions are persistent, an additional step such as centrifugation or re-extraction may be needed to break the emulsion and fully extract the analyte. These are costly analytical steps.

Another example where several initial or preliminary processes are required to chemically release, purify, and concentrate target analytes is the analysis of total fat. The steps involved includes: (i) hydrolysis in a hydrochloric acid (HCl) solution, (ii) removal of water soluble compounds in a bi-phase separation of an aqueous phase and organic solvent phase (in a Mojonnier flask), and (iii) evaporation of solvent for gravimetric quantitation of isolated fat. In other total fat methods, fat can be captured by oleophilic filters while allowing the aqueous solution to pass through. The residue and filter then must be thoroughly dried before extraction with organic solvents. The drying step removes trace water from the hydrolyzed sample which subsequently enables the non-polar solvent to penetrate the otherwise polar hydrolyzed sample. After extraction, the solvent containing the fat is evaporated and the isolated fat is quantitated gravimetrically. It will be appreciated that these methods are time-consuming, fiscally burdensome, and labor intensive.

In view of the difficulties and complexities of the current methods associated with the extraction of the 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.

SUMMARY

Systems and methods for extracting an analyte from a sample are disclosed. In one embodiment, the system includes a reaction chamber comprising a reaction vessel having a column for receiving the 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 the dissolved analyte, from the reaction vessel.

In one embodiment, a 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 sorbent is disposed in the purification vessel for retaining contaminants from the soluble components from the reaction mixture and passing a purified analyte.

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

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

DETAILED DESCRIPTION

The present disclosure is directed to an analyte extractor (or instrument) configured to automatically extract (i.e., isolate) analytes from complex matrices for subsequent quantitative analysis by, e.g., chromatography, spectrophotometry or gravimetric measurement. While the exemplary analyte extractor is principally configured to extract (i.e., isolate) fats and fat-soluble analytes, it should be appreciated that the device is equally applicable to any device having as its principle function, the liberation, extraction, purification and isolation of analytes which must be separated from complex matrices. Furthermore, while the exemplary analyte extractor includes a variety of chambers/vessels/containers/processes, in series, for isolating analytes for subsequent quantitative analysis, it will be appreciated that other embodiments may utilize fewer chambers/vessels/containers/processes to produce samples for further testing. For example, the analyte extractor may not utilize the evaporation chamber to produce an analyte for subsequent analysis. Moreover, while the exemplary instrument includes as many as four assay stations/positions a, b, c, d in parallel, i.e., in juxtaposed relation, for performing extraction processes on four (4) complex samples, it will be appreciated that the analyte extractor may utilize any number of assay stations or positions to extract analyte from samples.

FIG. 1depicts an exemplary analyte extractor10in accordance with the teachings of the present disclosure that includes a body or chassis12that allows for the fixed and detachable mounting of various components of the extractor10. The exemplary analyte extractor10comprises a plurality of vertically aligned chambers including a reaction chamber100, which is located above a purification chamber200, which is located above an evaporation chamber300for mounting one or more like components, i.e., components performing the same or similar operation.

Samples (e.g., food or feedstuff samples) can be deposited into and received by one or more reaction vessels104a,104b,104c,104din the reaction chamber100. As used herein, the term “vessel” generally refers to, e.g., a column, tube, etc. that can contain a fluid and allow the fluid to pass through. As used herein, the term column and tube are used interchangeably. A mixture of various solutions can be added to the samples in the reaction vessels104a,104b,104c,104dafter which agitating (e.g., mixing) can be performed, and heat can be added, to accomplish a first function where a dissolved analyte of the sample is produced in the soluble components of the reaction mixture. The dissolved analyte flows serially down through a filter, to one or more detachable purification vessels (e.g., columns, tubes, etc.)204a,204b,204c,204dassociated with the purification chamber200that are vertically aligned along same axis and below the respective reaction vessels104a,104b,104c,104d, such that a second operation can be performed to produce a purified analyte contained in solvent. Similarly, the purified analyte contained in solvent in the second row or purification chamber200can flow serially down to one or more containers (e.g., flasks)304a,304b,304c,304din yet another row associated with the evaporation chamber300that are vertically aligned along the same axis and below the respective purification vessels204a,204b,204c,204d, such that yet another operation can be performed.

In the described embodiment, the analyte extractor10may comprise a plurality of columns/stations/lanes a, b, c, d for integrating a plurality of vessels (e.g., columns, tubes, etc.) and containers (e.g., flasks) in parallel. As such, a plurality of samples, corresponding to the number of stations, can be processed simultaneously, vastly increasing throughput. Control inputs to the analyte extractor10may be made through a display, command, input or touch screen22. All variables associated with a method or a process may be input through the display/screen22. Each of these system components and method steps will be discussed in greater detail in the subsequent paragraphs.

FIGS. 2, 3 and 4depict detailed schematics of the exemplary analyte extractor10of the present disclosure depicting the relevant internal details and components of the instrument. More specifically, pumps P can be associated with valves V and flow meters M to inject a volume of liquid/solution into the reaction vessels104a,104b,104c,104dof the reaction chamber100. In one embodiment, a diffusing nozzle can be located at the inlets of the reaction vessels104a,104b,104c,104dto deflect the pumped solution toward an internal wall of the reaction vessel to wash down and dissolve analyte material disposed along the internal wall of the reaction vessel.

A heater H can be operative to heat the sample/mixture within one or more of the chambers100,300effectively forming an oven in each chamber100,300. A blower B can be operative to circulate air within one or more of the chambers100,300. One or more temperature sensors T1and T2control temperature in chambers100,300. Temperature sensors STmay also be provided in, on or integrated with, the reaction vessels104a,104b,104c,104dto provide temperature feedback in close proximity to, or within, the sample analyte being processed/evaluated.

One or more actuators A may be used to open/close a ganged shuttle valve150at appropriate intervals in the analyte extraction process. A plurality of reservoirs RL1, RL2, RL3, RL4and corresponding valves V may be employed for combining the various solutions with the sample within at least the reaction chamber100. Furthermore, while in the exemplary embodiment, each of the reservoirs RL1, RL2, RL3, RL4includes a valve V, it will be appreciated that a single valve V may be used to control the flow associated with two or more reservoirs RL1, RL2, RL3, RL4. A processor20, such as a microprocessor can be operative to control all processes within the system. Similarly, while a single processor20is shown to control the operations associated with each of the chambers,100,200,300, it will be appreciated that several microprocessors may be employed to control independent functions of the analyte extractor10. Finally, a power source (not shown) can be used to activate the pumps P, heater H, blower B, valves V, actuator A, temperature sensors ST, T1, and T2, pressure sensor SP, liquid sensors SL, ganged shuttle valve150and processor20of the analyte extractor10. Similarly, the pumps P, valves V, and flow meters M are operatively coupled to the processor20such that an accurate flow and quantity of solutions contained in fluid reservoirs RL1, RL2, RL3, RL4, may be supplied to the reaction vessels104a,104b,104c,104d.

Reaction Chamber

The analyte extractor10performs a variety of critical operations to isolate the target analytes from the complex sample. In the first process, i.e., in the reaction chamber100, a sample can be exposed to one or more solutions, while being agitated and heated in an oxygen-free environment. This step releases the target analyte from a complex matrix. Fat soluble vitamin analysis typically releases the vitamins through an ethanolic saponification reaction, while total fat analysis typically releases fat through a HCl hydrolysis reaction. These reactions result in an analyte which can be dissolved in a complex mixture of solutions along with an insoluble residue. This mixture can be filtered before it is passed onto the purification vessels (the second chamber200of the analyte extractor or instrument10).

The reaction vessel104has a unique design that allows for the separation of the liquid portion from the insoluble residue. To facilitate the discussion, a single reaction vessel104awill be described in detail with the understanding that adjacent reaction vessels104b,104c,104dcan be essentially identical and do not require or warrant additional description. Hence, when referring to one reaction vessel in the reaction chamber100and/or one component associated with the purification and evaporation chambers200,300, it should be understood that the vessel or step being described can apply to all of the same components associated with an adjacent assay station. The reaction vessel104acan be removable so that it can be placed on a balance/scale and sample can be weighed directly into the vessel104a.

The reaction vessel104acan be chemically inert and designed to withstand strong acids, bases, and organic solvents at temperatures ranging from, e.g., between 20° C. to 105° C. Prior to reaction, the reaction vessel104acan be closed (e.g., automatically closed) by lowering a chemically inert reaction vessel cap (i.e., lid, plug, top, etc.)108aover and enclosing the top opening of the vessel104a. Each reaction vessel cap108amay contain temperature and pressure sensors ST, SP, and apertures (i.e., ports, apertures, orifices, etc.)112afor vent- and liquid supply lines. The reaction vessel104acan 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 E, an oxygen-free environment can be created in the reaction vessel by inserting an inert gas, such as nitrogen (N2) gas.

Each reaction vessel104acan be detachable to facilitate removal, cleaning, sterilization and loading of sample material (not shown). The reaction vessel104acan comprise a cylindrically shaped reaction vessel column106amade of, e.g., borosilicate glass, a reaction vessel cap108ahaving a plurality of apertures112a, and a detachable reaction vessel base110a. It will be understood that the scope of the invention includes reaction vessels104ahaving a shape different than a cylindrical column. In addition to receiving temperature and pressure sensors ST, SP, the apertures112amay receive fluids or vent gases/volatiles from the reaction vessel104asample mixture. A liquid sensor SLmay be provided through the top or bottom of each vessel or container to ensure that liquids have drained or evaporated from the respective vessel or container. For example, inFIG. 4, the reaction vessel104amay include a liquid sensor SLthrough the detachable reaction vessel base110ato indicate when fluids have drained from the reaction vessel104a. Alternatively, or additionally, a liquid sensor SLmay be disposed in the flexible tubing between a reaction vessel drain (e.g., a drain)116aand the shuttle valve150. When the refractive index changes, the liquid sensor SLcan determine that liquid is no longer present in the tube, hence, no longer remaining in the reaction vessel104a.

While the exemplary borosilicate glass reaction vessel column106acan be configured to be cleaned, sanitized and reused, it will be appreciated that other materials may be employed which are disposable. For example, a transparent polypropylene cylindrical column may be employed with built-in components to facilitate rapid deployment and reuse of a reaction vessel104a. That is, a disposable reaction vessel104amay include the reaction vessel cap108ahaving apertures112afor receiving the temperature sensor ST, the pressure sensor SP, and one or more fluid fill lines. The disposable vessel104amay also include the reaction vessel base110aincluding a mixer driver132a, mixing stir bar130a, a requisite reaction vessel filter140, and the reaction vessel drain116a. An O-ring seal may or may not be required inasmuch as other sealing methods may be employed and the reaction vessel base110amay or may not be detachable.

In the described embodiment, the reaction vessel cap108acan be fixed to the instrument and be able to apply a downward force to seal the reaction vessel cap108a, the reaction vessel column106a, and the detachable reaction vessel base110aof the reaction vessel104a. The reaction vessel cap108aincludes at least two apertures112adisposed in fluid communication with at least one of the fluid reservoirs RL1, RL2, RL3, RL4, and at least one supply of nitrogen gas (N2). Nitrogen gas N2may be injected into the reaction vessel104ato produce an oxygen-free or oxygen-starved environment. As such, evaporating solvents cannot form a combustible gas inasmuch as the solvent-to-oxygen ratio can be maintained below combustion levels. The back pressure from the nitrogen gas (N2) can also function to facilitate flow of the dissolved analyte through the particulate reaction vessel reaction vessel filter140aand the reaction vessel drain116a.

The vessel caps108amay be fabricated from a plastic. In one embodiment, the plastic can be a polytetrafluoroethylene polymer, commonly known as a Teflon material (Teflon® is a registered Trademark of E.I. du Pont de Nemours and Company, located in Wilmington, State of Delaware). Since the vessel caps108aare exposed to large temperature variations, i.e., between room temperature to over one-hundred degrees Celsius (100° C.), the effect of thermal expansion must be considered to ensure a gas- and fluid-tight seal with the reaction vessel column106a. With respect to the reaction vessel cap108a, an elastomer or rubber O-ring can be sealed between the wall surface of the reaction vessel column106aand the reaction vessel cap108a. This arrangement and geometry can improve seal integrity since the sealing interface can be compressed due to the difference in thermal growth, or coefficient of thermal expansion, between the plastic vessel cap108aand the borosilicate glass reaction vessel column106a.

FIG. 5shows a detailed diagram of the reaction vessel base110a. The reaction vessel base110acan also be fabricated from a polytetrafluoroethylene polymer or plastic material (e.g., Teflon®). The O-ring seal118acan be disposed between the outer peripheral surface122aof the reaction vessel column106aand the inner wall of the reaction vessel base110a. Inasmuch as the growth of the plastic reaction vessel base110acould be greater than the borosilicate glass reaction vessel column106a, an outer ring or sleeve114aof reinforcing material may surround the plastic reaction vessel base110ato restrict its outward growth. The material selected for fabricating the outer ring or sleeve114ashould have a lower coefficient of thermal expansion than the plastic reaction vessel base110, and preferably, a similar rate of thermal expansion of the borosilicate glass. In the described embodiment, the outer ring or sleeve114acan be fabricated from a metal such as stainless steel. As a consequence, the seal integrity may be maintained or improved by an inwardly directed radial force124aapplied by the metal outer ring114a. To improve seal integrity yet further, the O-ring seal118ais received within a groove120ahaving a substantially dove-tailed cross-sectional geometry. This configuration captures the O-ring seal118aas the reaction vessel column106aslides into, and received within, the cavity of the plastic reaction vessel base110a. That is, the dove-tail groove120amaintains the efficacy of the sealing interface along the outer peripheral surface122aof the reaction vessel column106a, especially during assembly of the plastic reaction vessel base110a.

The reaction vessel104acan include a mixing stir bar130aand concentric mixer driver132a, which can be mounted below the reaction vessel base110a, and operative to mix fluids with the sample in the reaction vessel104a. More specifically, the mixing system comprises a mixing stir bar130aand a mixer driver132acoaxially aligned. The reaction vessel drain116aof the reaction vessel base110acan be aligned with and in fluid communication with an extraction port117aformed within a housing of the mixer driver132a. Furthermore, an O-ring134acan be disposed at the interface of the between the reaction vessel drain116aof the reaction vessel104aand the extraction port117aof the mixer driver132ato provide a fluid seal during operation. This design allows a drain to exit at the bottom of the reaction vessel base110a.

A high torque mixing system is preferred inasmuch as when combining certain samples and chemical solutions, reactions occur that significantly increase viscosity. In addition, the mixing system must be sufficiently robust to completely mix bi-phase solutions within a limited diameter vessel. The mixer motor has the ability to vary speed and direction, thus enabling the magnet to break free and spin under highly viscous conditions. The mixing stir bar130acan be magnetic, i.e., has a north and south pole, which repels or attracts relative to the poles produced by the magnetic mixer driver132a. More specifically, the mixer driver132acan define a torus-shaped electrical winding circumscribing the extraction port117aand creates an alternating magnetic flux field for driving the mixing stir bar130aabout a rotational axis. The mixing stir bar130aagitates the sample as one or more saponification fluids or solvents are added to the reaction vessel104a.

In the described embodiment, the mixer driver132aportion of the mixer130a,132acan be disposed below the reaction vessel104aand outside the reaction chamber100, which can form an oven when heated. As a consequence, the mixer driver132ais unaffected by the heat of the reaction chamber100. Additionally, the current-driven magnetic mixer driver132cannot produce an electrical spark in the reaction chamber100which may contain combustible gases as a consequence of the use of solvents, such as ethanol or hexane in the reaction chamber100.

The reaction vessel104acan be disassembled into a reaction vessel column106aand reaction vessel base110awhich allows for the placement of a reaction vessel reaction vessel filter140atherebetween. The compressive force exerted by the reaction vessel cap108aseals and secures the perimeter of the reaction vessel reaction vessel filter140a, such that particulates cannot circumvent the reaction vessel reaction vessel filter140a. The selective reaction vessel reaction vessel filter140ais capable of filtering insoluble particulate matter from the dissolved analyte material. More specifically, the reaction vessel reaction vessel filter140aseparates liquids from solids when performing a vitamin analysis. Furthermore, when performing a fat analysis, the reaction vessel reaction vessel filter140aquantitatively retains the lipid fractions while removing unwanted aqueous fractions to waste.

The reaction vessel reaction vessel filter140acan function as a temporary valve when the reaction vessel104ais removed from the reaction chamber100, filled with sample material, and weighed. That is, since the reaction vessel104amust contain a dry or wet sample while being loaded, weighed and, subsequently, reassembled into the reaction chamber100, the reaction vessel reaction vessel filter140aprevents the sample, or a portion of the sample, from escaping through the reaction vessel drain116a. The reaction vessel filter140amay vary in composition depending upon the chemical resistance properties and the type of analysis being performed.

For example, when performing a fat analysis on a food sample, the reaction vessel filter140acan be fabricated from a filter media having the capacity to retain particles two microns (2 μm) and larger. Typically, the reaction vessel filter140awill range from between approximately two microns (2 μm) to approximately fifteen microns (15 μm) when performing such analyses. When performing a vitamin analysis, the filter media of the reaction vessel filter140acan be fabricated from a filter material having a pore size less than approximately eight microns (8 μm). Typically, the filter media of the reaction vessel filter140awill range from between about eight microns (8 μm) to about thirty microns (30 μm). The retention of particulate when performing vitamin analyses does not need to be comprehensive. While it is important that fines do not clog fine tubing and valves, it is not critical that all fines are retained in the reaction vessel filter140abecause contrary to fat analysis, the entire saponified mixture of liquid and ultrafine particles are transferred to the purification vessel204a. The purification vessel204anot only retains the polar compounds but also filters out any fine particles that pass through the reaction vessel filter140a.

Shuttle Valve

It will be appreciated that while the reaction is occurring in the reaction vessel104a, the sample and dissolved analyte remain in the reaction chamber104afor a prescribed period (e.g., a dwell period). In one embodiment, a timer is provided to determine a dwell time associated with the operation of, e.g., the mixer, pump, and heat source and providing a dwell signal indicative of the operating time of each.

In the described embodiment, this can be accomplished by a shuttle valve150, which prevents the gravitational flow of the dissolved analyte from the reaction vessel104afor the prescribed dwell period.FIGS. 7A-7Cdepict schematic sectional views along the length and through each of the ports156a-156d,158a-158dof the shuttle valve150in different configurations of the shuttle valve150. Arrows7A-7A,7B-7B,7C-7C illustrate the direction of the cross-sectional plane and do not provide information concerning the kinematics of the shuttle valve operation. As will be appreciated upon review of the subsequent paragraphs, the plates152,154of the shuttle valve150slide orthogonally relative to the direction of arrows7A-7A,7B-7B,7C-7C.

InFIGS. 3 and 7A-7C, the shuttle valve150can comprise a pair of sliding plates, i.e., an upper or first plate152, and a lower or second plate154, wherein the first plate152includes ports156a,156b,156c,156dwhich are horizontally spaced in the plane of the plate152. The plates152,154are interposed between the reaction vessel drains116a,116b,116c,116dof the respective reaction vessels104a,104b,104c,104dand the input ports202a,202b,202c,202dof the respective purification vessels204a,204b,204c,204d.

Examination of configuration of the plates152,154shown inFIG. 7A(closed position) reveals that the ports156a,156b,156c,156dof the first plate152are dead-ended or closed against the upper surface of the second plate154. Accordingly, the shuttle valve150is in a closed position for inhibiting the passage of dissolved analyte from the reaction vessel drains116a,116b,116c,116dof the respective reaction vessels104a,104b,104c,104dto the input ports202a,202b,202c,202dof the respective purification vessels204a,204b,204c,204d.

Examination of configuration of the plates152,154shown inFIG. 7Breveals that shuttle valve150is in a open-to-waste position facilitating the passage of fluid through the first and second plates152,154by means of the aligned port pairs156a,157a,156b,157b,156c,157c,156d,157din plates152,154, respectively. That is, the actuator A moves the relative position of the plates152,154such that the ports156a,156b,156c,156dfrom one plate152align with the ports157a,157b,157c,157dof the opposing plate154. It will be appreciated that the input ports156a,156b,156c,156dare aligned with output ports157a,157b,157c,157dlocated on the lower plate154to allow the flow of fluid from the reaction vessels104a,104b,104c,104dacross the plates152,154towards a drain reservoir. This can be achieved by moving the second or lower plate154in one direction, e.g., in the direction of arrow L to the left (FIG. 7B), while maintaining the position of the upper plate152, i.e., held stationary. In another embodiment, the second or lower plate154can remain stationary while the first or upper plate152moves to the right. Potential uses of the drain-to-waste position are to remove solvent vapors from the reaction vessel or to remove unnecessary liquids from the reaction vessel.

Examination of configuration of the plates152,154shown inFIG. 7Creveals that shuttle valve150is in a open-to-vessel (purification) position facilitating the passage of fluid through the first and second plates152,154by means of the aligned port pairs156a,158a,156b,158b,156c,158c,156d,158din plates152,154, respectively. That is, the actuator A moves the relative position of the plates152,154such that the ports156a,156b,156c,156dfrom one plate152align with the output ports158a,158b,158c,158dof the opposing plate154. It will be appreciated that the input ports156a,156b,156c,156dare aligned with output ports158a,158b,158c,158dlocated on the lower plate154to allow the flow of dissolved analyte from the reaction vessels104a,104b,104c,104dacross the plates152,154to the respective purification vessels204a,204b,204c,204d. This can be achieved by moving the second or lower plate154in one direction, e.g., in the direction of arrow R to the right (FIG. 7C), while maintaining the position of the upper plate152, i.e., held stationary. In another embodiment, the second or lower plate154can remain stationary while the first or upper plate152moves to the left. Accordingly, the shuttle valve simultaneously controls the flow between the reaction vessels104a,104b,104c,104dand the purification vessels204a,204b,204c,204d.

The low-profile geometry of the shuttle valve150allows the valve150to be mounted below the reaction chamber100while remaining in close proximity to the reaction vessel drains116a,116b,116c,116dof the reaction vessels104a,104b,104c,104d. Moreover, the use of a small inner-diameter tubing to connect the reaction vessel drains116a,116b,116c,116dto the purification vessels204a,204b,204c,204densures a minimal air gap therebetween. This ensures that liquid does not migrate into the valve area when the reaction chamber100is operational. Finally, the shuttle valve150can be pneumatically actuated reducing the potential for electrical sparks in areas which may contain evaporated solvent and potentially flammable/combustible gases.

Purification Chamber

In the purification chamber200, the analyte extractor10separates wanted fractions of the dissolved analyte from reaction vessel104afrom unwanted fractions of the dissolved analyte from reaction vessel104by passing the dissolved analyte through a purification vessel204filled with a selective sorbent216a(e.g., a solid phase filter material, such as siliceous earth, also commonly referred to as diatomaceous earth (de) or other chromatographic media or aluminum oxide). Before passing the dissolved analyte through the purification vessel204, the selective sorbent can be conditioned to retain polar compounds in the purification vessel204while permitting the more non-polar target analytes to pass through the purification vessel204. This can be achieved by passing a specific quantity of water and ethanol through the selective sorbent, either prior to or simultaneously with the sample.

As shown inFIGS. 1, 3, and 6, the purification chamber200defines a cavity for mounting four (4) purification vessels204a,204b,204c,204dwhich can be arranged substantially horizontally across the purification chamber200. Continuing with the description above, a single purification vessel204awill be described with the understanding that adjacent vessels204b,204c,204dcan be essentially identical and do not require or warrant additional description. Hence, when referring to one purification vessel204ain the purification chamber200, it should be understood that the vessel being described applies to all of the vessels in the adjacent assay stations.

The purification vessel204acan be configured to receive the dissolved analyte from the reaction vessel104afollowing the reaction and filtration of the analyte in the reaction chambers100. More specifically, the purification vessel204acan be in fluid communication with the reaction vessel drain116aof the reaction vessel104athough the shuttle valve150.

The purification vessel204aincludes a polymer (e.g., polypropylene) cylindrically shaped purification vessel column208ahaving a purification vessel drain (e.g., a tapered nozzle)210aat one end and a top opening212aat the other end, equivalent in size to the diameter of the purification vessel column208a. It will be understood that the scope of the invention includes purification vessels204ahaving a shape different than a cylindrical column. While, in the disclosed embodiment, the purification vessel204acan be a polymer, it should also be understood that the purification vessel204acould be any flexible or rigid single use container.

The purification vessel column208acan be configured to receive: (i) lower purification vessel filter214afor being disposed above the purification vessel drain210a, (ii) a volume of a selective sorbent (e.g., Siliceous Earth (SE))216a, (iii) a purification vessel diffuser218a, and (iv) a purification vessel cap (i.e., lid, plug, top, etc.)220afor controlling the flow of dissolved analyte and nitrogen into the purification vessel column208a. The lower purification vessel filter214aand purification vessel diffuser218aare employed to hold the selective sorbent216awithout allowing any of the sorbent through the purification vessel filter214aor the purification vessel diffuser218a. The pores of the lower purification vessel filter214aand the purification vessel diffuser218amust be sufficiently small to hold the selective sorbent material.

The dissolved analyte from the reaction vessel104aenters the purification vessel column208avia the reaction vessel cap apertures224ain the purification vessel cap220a. In one embodiment and as shown inFIG. 3, an input port202acan feed the mixture from the reaction vessel drain116ato the purification vessel cap220aof the purification vessel column208a. Once through the reaction vessel cap aperture224a, the purification vessel diffuser218adiffuses or spreads the analyte solution to prevent the formation of flow channels, (similar to erosion caused by running water) through the selective sorbent216a. As such the analyte material is spread in a substantially uniform manner over the top of the selective sorbent216a. Once the dissolved analyte from the reaction vessel104ais passed through the purification vessel204a, the purified analyte contained in solvent flows serially through the purification vessel drain210ato one or more containers (e.g., flasks)304a,304b,304c,304din the evaporation chamber300.

Evaporation Chamber to Evaporate Liquids from the Purified Analyte Material

In the evaporation chamber300, the solvent can be evaporated from the purified analyte such that the purified analyte may be collected for subsequent quantitation (e.g., by HPLC or GC). InFIG. 4, the evaporation container (e.g., flask)304ais in fluid communication with, and receives, the purified analyte material from the purification vessel204a, i.e., from the purification vessel drain210a. It will be understood that the scope of the invention includes evaporation containers304a-304dhaving a shape different than a flask. The purified analyte mixture contains solvents which are evaporated in an oxygen free environment within the evaporation container304a. That is, the evaporation container304acan be filled with a inert gas (e.g., nitrogen N2) via a nozzle308a. The nozzle308acan be disposed in combination with a cap (not shown) inserted within the opening of the evaporation container304a, while an exhaust aperture in the cap (not shown) allows the high velocity flow of inert nitrogen gas (N2) to move the solvent within the container304aand promote evaporation.

In addition to movement of the solvent within the evaporation container304a, the evaporation container304acan be heated to increase the rate of evaporation. The container304acan be continuously purged with nitrogen to protect the analyte from oxidation. To protect light sensitive analytes from select wavelengths of ultraviolet light, UV protected polycarbonate doors can cover chambers100and300.

InFIGS. 2 and 4, the ducting from the heater H can be bifurcated such that a flow of heated air can be directed to both the reaction vessels104a-104din the reaction chamber100and the evaporation containers304a-304din the evaporation chamber300. Temperature sensors T1, T2, ST, located in the reaction and evaporation chambers100,300, provide temperature signals to the processor20. These signals are indicative of the instantaneous temperatures within each of the chambers100,300and within each of the reaction vessels104a,104b,104c,104dand each of the evaporation containers304a,304b,304c, and304d. The processor20can compare these signals to predefined temperature values stored in processor memory. The processor20evaluates the difference or error signal between the stored temperature value and the actual/instantaneous temperature to raise or lower the temperature in the respective chambers100,300and/or reaction vessels104a,104b,104c,104dand evaporation containers304a,304b,304c, and304d.

Temperature sensors T1, T2, and STcan be located in various locations within the analyte extractor10for the purpose of mapping the temperature in the reaction and evaporation chambers100,300. With respect to the reaction chamber100, the described embodiment shows a temperature sensor T1to determine the temperature within the chamber100while a temperature sensor STmeasures the temperature in each of the upper end caps to obtain a temperature reading from within each of the reaction vessels104a,104b,104c,104d. While these locations provide a reasonably accurate picture of the temperature within the reaction chamber100and within the vessels104a,104b,104c,104d, it will be understood that other locations may provide more direct or accurate temperature measurements.

For example, in one alternate embodiment, a thermocouple can be attached to the reaction vessel column106aof each of the vessels104a,104b,104c,104dsuch that the temperature of the sample mixture can be measured within the respective vessels104a,104b,104c,104d. This may be done assuming, of course, that the 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 base110aof each of the vessels104a,104b,104c,104dsuch that temperature can be measured at the bottom of the respective vessels104a,104b,104c,104d.

The fluid reservoirs RL1, RL2, RL3, RL4may contain one or more strong basic or acidic fluids, such as potassium hydroxide (KOH) or hydrochloric acid (HCl). Alternatively, the reservoirs RL1, RL2, RL3, RL4may contain one or more solvents including, water (H2O), ethanol (CH3CH2OH) and hexane (CH3(CH2)4CH3). Flow from the reservoirs RL1, RL2, RL3, RL4, and/or from the nitrogen supply may be supplied or activated by the external pumps P and/or controlled by one or more valves V and/or flow meters M. In addition to the apertures112afor accommodating fluid or gaseous flow, the reaction vessel cap108amay include at least one aperture for accepting a pressure sensor SP.

The processor or controller20can be responsive to the temperature, pressure, and liquid sensor signals ST, SP, SLprovided by each of these sensors for changing the temperature, pressure, and flow within the reaction chamber100, purification chamber200, and evaporation chamber300. With respect to the temperature in the reaction chamber100, an alternate or second temperature sensor T1(seeFIG. 2) may be disposed in the reaction chamber100rather than through the reaction vessel cap108aof the reaction vessel104a. The temperature in the reaction chamber100may be varied by controlling the output of the heater H and the blower B. Accordingly, the processor20can be responsive to temperature signals from one or more temperature sensors.

Temperature sensor T1can vary or change the output of the heater H along with the flow of the blower B. In one embodiment, a heat exchanger can be connected to the heater H and the blower can direct air over the heat exchanger to create heated air. It will be appreciated that the flow of heated air can be bifurcated such that while performing or causing the reaction, some or all of the heated flow HScan be directed to the reaction chamber100and, during evaporation of the liquid(s) contained in the sample, some or all of the flow HEcan be directed to the evaporation chamber300. Consequently, the processor20may direct the flow from the heater H to either of chambers100,300via a bifurcated duct (BD). The processor20may be responsive to the pressure signals to increase the pressure of the nitrogen gas (N2) during the reaction to improve or increase the flow rate through of the reaction vessel drain116a. This can also serve as a method to inject nitrogen gas (N2) into the subsequent purification chamber200, following the reaction in the reaction chamber100.

Designing an automated system and method was a complex endeavor involving a series of inventive steps that were not obvious at the start of the project. A number of significant difficulties and challenges were overcome to develop an instrument that performed automatically.

The reactions necessary to chemically release the analyte produces a reaction mixture that is not always compatible with the next process. For example, the requirements for conditioning a SPE (solid phase extraction) column are not typically compatible with a reaction mixture designed to chemically release the analyte. The passage of the analyte from the reaction chamber, with both solid and liquid fractions, was not compatible with a valve function necessary to transfer the analyte to the next step. The solution was to carry out the reaction prior to, or above, filtration so that only the liquid would pass through under certain conditions. It was then discovered that changes to the reaction and a specialized filtration design was required. More specifically, unique filter designs, special mixing configurations and changes in solutions were required.

For example, the solution that passed through the filter contained a complex mixture of the analyte and contaminants in an aqueous and organic solvent solution. In order to isolate the analyte, an SPE column was employed. The stationary phase of the SPE was capable of retaining the contaminants, water and other polar solvents, while allowing the non-polar solvents (e.g., hexane) to elute the analyte for transfer to the evaporation chamber. The resulting system included a reaction vessel having a bottom portion configured to be detachably released to facilitate filter removal/replacement and sample introduction. Furthermore, the valve system was developed that facilitated transfer of solution to the next chamber or to waste. Finally, an SPE column was employed for purification, which communicated with a flask in an evaporation chamber that removed the solvent by nitrogen gas together with a directed heat input.

Examples of Analyte Extraction

Vitamins

The analyte extractor10of the present disclosure is capable of extracting analytes from complex matrices. One example is that of the extraction of vitamins A and E from infant formula. Infant formula can be reconstituted with water, after which a sub-sample (aliquot) is weighed in the reaction vessels104a,104b,104cand104d, along with a combination of antioxidants. Each of the reaction vessels104a,104b,104cand104dcan be subsequently assembled into the reaction chamber100. The reaction vessel caps108a,108b,108c,108dfor each can be combined with the respective reaction vessels104a,104b,104c,104d(i.e., effecting a fluid-tight seal with each of the reaction vessels104a,104b,104c,104dvia the downward force applied by the mounting bracket) once the reaction vessels104a,104b,104c,104dis placed on the instrument10. The following processes are automatically controlled by the processor20: addition of saponification solutions (KOH and ethanol), mixing and heating to 75° C. for 30 minutes, the addition of water, cooling to 60° C., passing the reaction mixture through the filter and allowing the liquid to pass into the SPE column containing diatomaceous earth, eluting vitamin A and E from the SPE column with hexane (leaving the contaminants behind) and passing vitamins into the round bottom flasks in the evaporation chamber300, the solvent is then evaporated by a vigorous flow of nitrogen and a heat source focused on the bottom of the flask. The isolated oils containing vitamins A and E are manually re-dissolved in hexane and injected into an HPLC for quantitation.

Total Fat Analysis

Another example is that of total fat analysis, through acid hydrolysis. The analyte extractor10recovers total fat by combining the digestion (HCl) and extraction processes in the reaction chamber100with the separation capabilities of SPE. Ethanol can be used to displace the residual water and bridge the polarity gap with hexane, allowing the hexane to penetrate the sample residue and filter, ultimately dissolving the fat. The constant agitation coupled with heated solvent greatly enhances extraction ability. The SPE column binds the aqueous/ethanolic solvent and components and allows the hexane to elute with the fat. This selective capability of the SPE allows the analyte extractor10to bypass the traditional drying step of the hydrolyzed sample such that the total fat analysis can be performed in a single device/instrument.

The steps involved for analysis of total fat begins with sample breakdown in the reaction vessel104a. Before the sample is added, the selective, multi-layered reaction vessel filter140acan be placed in the reaction vessel104aover the reaction vessel drain116a. The reaction vessel reaction vessel filter140acomprises a combination of rigid and flexible layers which provide structural and loft benefits. Loft prevents the sample from clogging the filter during filtration, while the rigidity of the reaction vessel reaction vessel filter140aprevents it from moving under the reaction vessel column106a. The sample can be then added and reassembled into the mounting bracket within the reaction chamber100. In a total fat analysis, Hydrochloric acid (HCl) can be contained in one of the reservoirs RL1, RL2, RL3, RL4and can be automatically added into the reaction vessel104a.

Enhanced by continuous mixing, the sample can be heated in the HCl solution to release the bound fat. Once the process is complete, the aqueous solution can be filtered to waste through the shuttle valve150. By mixing and heating the sample in the reaction chamber100, chemical breakdown of the sample can be optimized, and fat completely released from the sample matrix. The chemical breakdown also reduces the formation of gelatinous materials which can clog a filter. Inasmuch as the chemical bonds are broken and contaminants are removed, the analyte extractor10can filter large samples through a relatively small filter allowing only the aqueous solution to pass.

The analyte extractor10bypasses the drying step by the integration of the SPE column. Rather than drying the sample, as discussed in the background section of this disclosure, the analyte extractor10automatically adds solvents (e.g., ethanol and hexane) to the sample residue remaining on the reaction vessel reaction vessel filter140a. The hydrophilic nature of the ethanol combines with the water to make a new solvent that can be compatible with the hexane enabling the hexane to extract the fat. After extracting the wet residue with solvent, the extracted solvent contains a dissolved mixture of medium-polarity substances along with the fat.

Therefore, the fat must be isolated from the nonfat components. In the present disclosure, this step can be performed by the SPE column, where polar and medium polar contaminants are separated from the non-polar, fat components. Furthermore, the SPE column interacts with the mixed sample solution allowing only non-polar solvents containing fat to exit the SPE column into the evaporation flask. Once the solvent and fat have entered the evaporation flask, the solvent is evaporated leaving only fat behind for further analysis.

The analyte extractor10is unique by comparison to other total fat analysis methods. For example, the analyte extractor10can perform total fat analyses with a single instrument which does not require a separate drying step. This differentiates the analyte extractor10from other extraction methods, which require the combination of at least two instruments and an oven drying step to accomplish total fat analysis.