Rapid sample preparation for analytical analysis using dispersive energized extraction

Abstract

An extraction method for preparing samples for analytical analysis is disclosed. The method includes the steps of placing a sample matrix containing one or more analytes in a heat conductive sample cup, positioning the heat conductive sample cup in a pressure-resistant reaction chamber, dispensing solvent into the heat conductive sample cup, dispersing the solvent and the sample matrix in the sample cup in the reaction chamber, heating the sample matrix and the extraction solvent in the heat conductive sample cup in the reaction chamber to a temperature at which the dispensed solvent generates an above-atmospheric pressure, and releasing the extraction solvent extract from the sample cup at atmospheric pressure.

Claims

1. An extraction method for preparing samples for analytical analysis comprising the steps of: placing a sample matrix containing one or more analytes in a heat conductive sample cup, said sample cup having an open mouth at one end and a partially open floor at the opposite end; positioning the heat conductive sample cup in a pressure-resistant reaction chamber; dispensing extraction solvent into the heat conductive sample cup that is inside of the reaction chamber, wherein said dispensing is carried out from positions selected from the group consisting of the open mouth of the sample cup, the partially open floor of the sample cup, and the open mouth and partially open floor of the sample cup; heating the sample matrix and the extraction solvent in the heat conductive sample cup in the reaction chamber to a temperature at which the dispensed solvent generates an above-atmospheric pressure; and releasing the extraction solvent extract from the sample cup at atmospheric pressure in which the above-atmospheric pressure from the heated solvent drives the release of the solvent extract.

2. An extraction method according to claim 1 further comprising: releasing the solvent extract into a cooling coil that has a length sufficient to reduce the temperature of the solvent extract to ambient or near-ambient temperature in the coil; and collecting the solvent extract from the coil.

3. An extraction method according to claim 1 wherein the partially open floor supports a filter or filter media and allows solvent extract to drain from the sample cup.

4. An extraction method according to claim 1 further comprising bubbling gas that is inert to the sample matrix through the bottom of said sample cup.

5. An extraction method according to claim 1 further comprising applying ultrasonic agitation during the pressurized heating step.

6. A method according to claim 1 further comprising conductively heating the reaction chamber to a temperature of between about 90 C. and 180 C. and generating resulting pressures of between about 50 and 250 psi (between about 345 kPa to about 1724 kPA).

7. An extraction method according to claim 1 wherein the extraction solvent is selected from the group consisting of water, weak acids, weak bases, ethyl acetate, methyl tertiary-butyl ether (MTBE), methylene chloride, hexane, acetone, hexane 2-propanol, cyclohexane, acetonitrile, methanol and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of some of the elements used to carry out the method of the invention.

(2) FIG. 2 is an exemplary full scan chromatogram of the BNA CMR extraction based on Example 1.

(3) FIG. 3 is an overlay of the Example 1 extraction according to the invention as compared to an ASE extraction

(4) FIG. 4 is a sample full scan chromatogram of the Example 2 polyethylene CRM extraction using the invention.

DETAILED DESCRIPTION

(5) A number of terms are used herein to describe the method.

(6) The term solvent is used in its well understood chemical sense; e.g., a substance capable of dissolving another substance (solute) to form a uniformly dispersed mixture (solution) at the molecular or ionic size level. The adjective organic is used in its well understood sense to embrace all compounds of carbon other than certain small molecule combinations of carbon with oxygen, sulfur, and metals, and in some cases halogens. See, Lewis, HAWLEY'S CONDENSED CHEMICAL DICTIONARY, 15th Edition, 2007, John Wiley & Sons

(7) The sample matrix is the material to be tested for the presence and optionally the amount of analyte.

(8) The analyte is the molecular compound of interest. As used herein, analyte can include samples with a plurality of analytes within a single sample.

(9) The solvent extract is the solution of analyte in a solvent following extraction.

(10) The sample cup is the container for the sample matrix and the solvent.

(11) The collection vessel is the container that collects the solvent extract following cooling.

(12) In a first aspect the invention is an extraction method for preparing samples for molecular analysis. In the method an extraction solvent and a sample matrix are placed into a sample cup, and the sample cup is positioned in a pressure resistant heating chamber. Typical (but not limiting) sample matrices include food, food packaging, and soil.

(13) As recognized by the skilled person (e.g., US EPA Method 3545) samples should be extracted using a solvent system that gives optimum reproducible recovery of the analytes of interest from the sample matrix, at the concentrations of interest. The choice of extraction solvent depends on the analytes of interest and no single solvent is universally applicable to all analytes.

(14) Typical (but not limiting) solid-liquid extraction solvents for molecular analysis include water, weak acids, weak bases, acetone, hexane 2-propanol, cyclohexane, dichloromethane, acetonitrile, methanol, and mixtures thereof.

(15) As set forth further herein, and without being bound by theory, it appears that the step of heating the reaction chamber to in turn heat the sample cup creates a sufficient pre-equilibrium thermal gradient to assist in mixing and agitating the solvent and the sample. In some embodiments the reaction chamber is pre-pressurized (about 25 psi has been found to be sufficient) to enhance the extraction and potentially enhance the pre-equilibrium thermal gradient and its potential benefits.

(16) In the method of the invention, the sample matrix can also be described as loosely packed in the sample cup. Although the term loose is likewise relative, it is used here in its normal sense as being free from anything that binds or restrains and free or released from fastening or attachment (Urdang, THE RANDOM HOUSE COLLEGE DICTIONARY, Random House Inc. (1972)). Because the sample matrix is loose, the addition of solvent from the top, the bottom, or both, helps disperse the sample matrix in the solvent.

(17) The extraction solvent and the sample matrix can also be mixed in the cup in the chamber using an agitating flow of a gas that is otherwise inert to the sample matrix, the analyte or the solvent. Those skilled in the extraction art will recognize that the gas can accordingly be selected based on the known parameters, and that in some cases compressed air will be appropriate while in others nitrogen or hydrogen may be best (with care based upon hydrogen's flammable characteristics). In other cases one of the noble gases (e.g., helium, argon) may be best.

(18) Other mixing techniques can be used (e.g., magnetic stirrers or other mechanical devices), but tend to require more complex instrumentation.

(19) The sample matrix and the solvent are then heated in the sample cup in the reaction chamber to a temperature at which evaporated solvent generates an above-atmospheric pressure. A temperature of 90 C.-180 C. is exemplary (the US Environmental Protection Agency suggests 120 C. for soil), at which temperature typical organic extraction solvents generate a corresponding pressure of 50-250 pounds per square inch (psi). In experiments to date, the time to reach this temperature is about 90 seconds, at which point extraction is substantially complete (it being understood that extraction is an equilibrium process). The pressure generated by the vapor from the solvent is then used to drain the solvent extract from the sample cup into a cooling coil that has a length sufficient to reduce the temperature of the extract to near ambient (e.g., 25 C.) while the solvent extract is in the coil. The solvent extract is then collected from the coil, typically in a collection vessel. In exemplary experiments, metal tubing with a length of about 10 feet tends to provide a dwell time of about 30 seconds, which is sufficient to cool the solvent extract to ambient or near-ambient temperature. Thus, the coil is typically used for space saving purposes, but a coil shape is optional rather than mandatory.

(20) The sample matrix and the extraction solvent can be added in amounts that are typical in this field. For example, a solid matrix is collected in a manner that provides between about 5 or 10 grams (g) of the sample matrix of interest. The amount of extraction solvent will be proportional; typically about 30-100 milliliters (mL).

(21) FIG. 1 is a schematic diagram of the basic elements of an instrument to carry out the method steps of the present invention.

(22) FIG. 1 illustrates a number of the features of the method in the context of a schematic diagram. FIG. 1 illustrates a heat-conductive, pressure-resistant sample cup 10 surrounded by a pressure resistant reaction chamber 12.

(23) In the context of the invention, a typical sample cup is a cylinder form of a heat-conductive material. Because the sample cup 10 is inside the reaction chamber 12, it experiences little or no differential pressure, and thus its mass can be minimized to encourage thermal transfer. In current embodiments, an aluminum cylinder about 3.5 inches (8.9 cm) long and about 1.25 inches (3.2 cm) in diameter, with a wall thickness of about 0.1 inches (2.54 mm) has been found to be appropriate. The terms heat conductive or thermally conductive are used herein in their well-understood sense to represent materials through which heat passes relatively quickly. The opposite is, of course, the term insulating, which is likewise well-understood as describing materials through which heat passes more slowly. On that basis, many metals and alloys are particularly useful for the vessel given that such conductivity is one of the distinguishing characteristics of most metals and alloys. Alternatively, many polymeric materials are considered insulating and ordinarily less helpful in the context of the invention. The thermal conductivities of many metals and alloys are published and widely disseminated, and an appropriate metal or alloy can be selected by the skilled person without undue experimentation.

(24) An appropriate sample cup is a cylinder that has an open mouth at one end and a partially open floor at or near the opposite end. Small changes in shape or position (i.e. of the cup, its mouth, or the open end) are, of course, within the expected scope of the invention. The partially open floor can support a filter or filter media and allow solvent extract to drain from the sample cup. The solvent can be dispensed into the sample cup from the top of the sample cup, through the bottom of the sample cup, or both.

(25) The combination of extraction solvent and the sample matrix that contains an analyte (schematically diagrammed by the horizontal lines 11) are maintained in the sample cup 10 using the one open and filtered end 13. The filter medium is designated at 14.

(26) FIG. 1 also shows that additional extraction solvent optionally can be added to the reaction chamber 12 outside of the sample cup 10 as indicated by the dotted line 15 to jacket the sample cup 10. A closure 46 seals the sample cup 10 in the reaction vessel 12.

(27) A heater 16 heats the solvent 15 in the reaction chamber 12 outside of the sample cup 10 to in turn heat the sample cup 10, the sample matrix 11 and the extraction solvent until the temperature generates an above-atmospheric pressure that together with the increased temperature drives the analyte substantially from the sample matrix into the extraction solvent.

(28) The solvent extract is then released by opening the reaction chamber to atmospheric pressure at the open end (e. g., using valve 21) so that the solvent extract can travel to a cooling tube 17 which has a length sufficient to cool the solvent extract to ambient or near-ambient temperatures so that the cooled solvent extract can be collected ready for analysis, for example in a collection vessel 20.

(29) In carrying out preparation of a sample for molecular analysis, the sample matrix is placed in the sample cup 10 which is then placed in the thermally conductive reaction chamber 12. A solvent from a supply 22 is delivered to the sample cup 10 (and thus to the sample matrix) through the valve 33, the associated passageways 24 and 25, and the delivery head 26. A liquid matrix sample can be delivered from a syringe pump 27, 30 and the valve 33. Additionally, solvent can be added to the bottom of the reaction chamber 12 using the valve 33, the line 28, the valve 21, and the line 31.

(30) FIG. 1 also illustrates that the gas agitation is carried out by delivering an inert gas from a supply 37 to a position at or near the bottom of the sample cup using the passageways 40 and 41, as controlled by the valve 42. If a secondary agitation is required, it can be carried out with a device such as the ultrasonic generator 43 which would typically be a piezoelectric transducer.

(31) The draining step takes place when the valve 21 is opened to atmospheric pressure so that the pressurized solvent vapor in the thermally conductive chamber 12 pushes the liquid solvent extract out through the passageway 31, then through the valve 21, and then cooling coil 17. The cooling coil is connected to a collection vessel 20 by the collection tube 32.

(32) Further to FIG. 1 and to complete the description of the possibilities, solvent can flow from the solvent supply 22 to the rotary valve 30 through the line 24. The line 47 connects the rotary valve 30 with the auxiliary valve 33. The line 28 connects the auxiliary valve 33 to the gas valve 21 which in turn can use the line 31 to deliver solvent to the bottom of the reaction chamber 12.

(33) The line 48 connects the rotary valve 30 to the syringe 40 so that liquids from the supply 22 can be metered into the syringe 40 from the supply 22 and thereafter from the syringe 40 into the sample cup and through the lines 35 to 25 and the dispenser head 26. The dotted line 15 represents the position of solvent between the sample cup 10 and the reaction chamber 12 when the solvent is used to jacket the sample cup 10.

(34) The gas supply 37 can supply extra pressure to the headspace through the lines 50 and 47 which, along with the gas flow to several other items, is controlled by the valve 32. The line 51 joins the valve 32 to the vent 35.

(35) As part of the gas pressure monitoring, the line 52 connects the valve 32 to the pressure gauge 23 and the pressure gauge 23 is wired to the processor 38 through the communication line 53. The processor 38 is also connected to the thermocouple 44 using the communication line 54 so that monitored combinations of temperature and vapor pressure for various sample extractions can be used to develop helpful standardized information.

(36) In order to provide agitating gas into the bottom of the reaction chamber 12 and the sample cup 10, the gas supply at 37 is also connected to the valve 21 through an appropriate line or tube 184.

(37) A pressure head seal 46 seals the sample cup in the reaction chamber. Line 56 drains solvent from valve 21 to the coil 17, and line 32 drains the coil 17 to the collection vessel 46.

(38) The nature of the method is such that it can be expressed in some additional aspects. In a second aspect, the steps include placing an extraction solvent and the sample matrix containing the analyte into a sample cup. Thereafter, the sample matrix and the extraction solvent are agitated, heated, and pressurized in the sample cup to extract the analyte from the heated sample matrix and into the heated extraction solvent. The pressurized heated extraction solvent extract is then drained at atmospheric pressure from the sample cup and through the cooling coil until the drained extraction solvent extract approaches or reaches ambient temperature. The cooled extraction solvent extract is then collected for analysis.

(39) In FIG. 1 the headspace in the sample cup 10 above the solvent 11 can be pressurized from the gas source 37 using the valve 32 and the line 47.

(40) Obviously, a wide ranging selection is available to the skilled person, and because the invention uses the same solvents and stationary phases as other methods, appropriate choices can be made without undue experimentation.

(41) If a second agitation step is needed, it can be carried out before, or concurrently with, the heating and pressurizing steps, and typically using ultrasonic vibration. Alternatively (or additionally) agitation can be carried out by feeding a gas that is inert to the solvent and the analyte.

(42) As in the previous embodiments, the step of draining the release solvent includes draining the heated release solvent in a coil that has a length sufficient to cool the drained release solvent to approach or reaching ambient temperature while the release solvent is in the coil. At that point, the release solvent containing the analyte is at a temperature ready for molecular analysis in conventional equipment.

(43) Basically, the method of the invention is appropriate for preparing any analyte that is stable at the expected temperatures and pressures.

(44) In each embodiment, solvents can be selected from the group consisting of water, weak acids, weak bases, ethyl acetate, methyl tertiary-butyl ether (MTBE), methylene chloride, hexane, acetone, hexane 2-propanol, cyclohexane, acetonitrile, methanol and mixtures thereof, but are not limited to that particular group.

(45) Each embodiment can use an ultrasonic second agitation step during the pressurized heating step.

(46) In each embodiment, the release of the solvent extract to atmospheric pressure is used to drive the solvent extract from the sample cup and into the cooling coil.

(47) In each embodiment, representative heating temperatures are 90-180 C. and representative resulting pressures are between about 50 and 250 psi.

(48) In yet another aspect, the invention can be expressed as the heated pressurized agitated mixture of an extraction solvent and a sample matrix containing an analyte in a sample cup.

EXPERIMENTAL

Example 1

(49) TABLE-US-00001 TABLE 1 Environmental Application; Extraction of BNA's from soil Sample Vol- Temper- Pres- Size Solvent ume Time ature sure Method (g) (1:1 v/v) (mL) (minutes) ( C.) (psi) Soxhlet 10 Hexane/ 150 1440 100 N/A Acetone Example 5 Hexane/ 30 5 100 <350 1 Acetone ASE 5 Dichloromethane/ 50 26 100 1500 Acetone

(50) Table 1 plots data from the extraction of bases neutrals and acids (BNA's) from soil comparing Soxhlet (EPA 3540C), the current invention (Example 1), and accelerated solvent extraction (ASE; EPA 3545). The volume and time for the indicated ASE's are taken from a run using the parameters set forth in Dionex application note 317. Analysis was carried out using gas chromatography followed by mass spectroscopy (GCMS; EPA 8270).

(51) The method of the invention uses significantly less solvent and takes significantly less time than the other methods. In particular, the preparation of the ASE extraction cell is generally tedious with over 10 components and steps, whereas the present invention uses just three straightforward pieces. On average, preparation of an ASE extraction cell takes about 15 minutes, while the invention is ready in a few seconds.

(52) TABLE-US-00002 TABLE 2 Environmental Application: Extraction of BNA's from soil; CRM Recovery Data (%) Analyte Proficiency Phenol 60.0 Hexachloroethane 51.1 Analyte Soxhlet % Proficiency Soxhlet Phenol 48.9 81.5 Hexachloroethane 38.6 75.5 Analyte ASE % Proficiency ASE Phenol N/A N/A Hexachloroethane 32.2 63 Analyte Invention % Proficiency Invention Phenol 60.2 100 Hexachloroethane 45.6 89.2

(53) Table 2 summarizes the data by percentage for BNA's in certified reference material (CRM) soil obtained from Waters Corporation (Milford, Mass. 01757 U.S.A.; ERA catalog number 727). As understood by those in the art, the goal is to obtain 100% recovery of the materials known to be present in the CRM sample. For each method, all of the recoveries were within the quality control performance acceptance limits, but the invention (Example 1) recovered all 39 analytes, while ASE recovered only 38, and failed to identify 2-methylnaphthalene. The invention accordingly had the best overall performance in terms of the analytes recovered and the percent recovery of the analytes.

(54) FIG. 2 is an exemplary full scan chromatogram of the BNA CMR extraction based on Example 1.

(55) FIG. 3 is an overlay of the Example 1 extraction according to the invention as compared to the ASE extraction. Each of the higher peaks represents the Example 1 extraction which significantly outperformed ASE in recovery. Additionally, the absence of an ASE peak at retention time 10.36 (2-methylnaphthalene) demonstrated the failure of ASE to identify this analyte.

Example 2

(56) TABLE-US-00003 TABLE 3 Extraction of Phthalates from Polyethylene Vol- Temper- Pres- Sample Solvent ume Time ature sure Method Size (g) (70:30 v/v) (mL) (minutes) C. psi Soxhlet 0.5 Acetone/ 150 1440 100 N/A Cyclohexane Example 0.5 Acetone/ 30 10 140 <350 2 Cylcohexane ASE 0.5 Hexane 50 63 120 1500

(57) Table 3 is a comparison chart as between Soxhlet, the invention (Example 2), and ASE for the extraction of phthalates from polyethylene. The volume and time for ASE are from a run using the parameters stated in a Dionex publication (Knowles, D; Dorich, B; Carlson, R; Murphy, B; Francis, E; Peterson, J, Richter, B. Extraction of Phthalates from Solid Liquid Matrices, Dionex Corporation, 2011) and all methods were based off of CPSC-CH-C1001-09.1 (Consumer Products Safety Commission, Test Method: CPSC-CH-C1001-09.3 Standard Operating Procedure for Determination of Phthalates; http://www.cpsc.gov/about/cpsia/CPSC-CH-C1001-09.3.pdf).

(58) Again, the method of the invention (Example 2) used significantly less solvent and took significantly less time than the other methods.

Example 2

(59) TABLE-US-00004 TABLE 4 CRM Recovery Data (%) % % Soxh- Example Soxhlet % Exam- let 2 w/ Example Soxh- ple Exam- Agita- 2 w/ let Analyte Soxhlet 2 ple 2 tion Agitation ASE ASE Bis (2- 72.6 57.7 79 73.4 101 24.3 33 ethylhexyl) phthalate Di-n-octyl 85.5 68.2 80 80.7 94 31.4 37 phthalate

(60) Table 4 compares the recovery data by percentage for extraction of phthalates from polyethylene in a CRM sample (SPEX CertiPrep CRM-PE001; Metuchen, N.J. 08840, USA). In this experiment agitation was carried out with 30 seconds of both bubbling and sonication prior to heating. Again, the invention (Example 2) recovery data was significantly better than ASE and showed an improvement with the use of agitation. Example 2's results with agitation match Soxhlet data which is considered the gold standard for extraction. All analytes in the CRM were recovered for all methods.

(61) FIG. 4 is a sample full scan chromatogram of the Example 2 polyethylene CRM extraction using the invention.

(62) In the drawings and specification there has been set forth a preferred embodiment of the invention, and although specific terms have been employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being defined in the claims.