ES2368274B2 - IMPROVED MINIATURIZED EXTRACTION DEVICE. - Google Patents

Abstract

Improved miniaturized extraction device. # An improved miniaturized device has been developed that allows the extraction with liquids at high pressures and controlled temperature of chemical species of different nature from semi-solid or solid samples packed in a closed cell, overcoming some limitations related to the thermal inertia of the system. The device incorporates a resistance inserted in a steel tube and an engine that guides air through the tube for heating the chamber where the extraction cells are placed. Both the resistors and the motor can be turned on and off independently. # The improved device allows you to shorten the time required to perform the extraction with liquids of chemical species of different nature from semi-solid or solid samples, which constitutes a very useful advance for the resolution of various problems within the field of sample preparation in Analytical Chemistry.

Description

Enhanced miniaturized extraction device.

Object of the invention

The present invention falls within the technical field of sample pretreatment for the analysis of chemical species in complex matrices.

A miniaturized device has been improved that allows the extraction with solvents at high pressures and temperatures of solid and semi-solid matrices, reducing both the time required for extraction at high temperatures and the waiting time between samples. The use of this new device allows a significant reduction in the overall sample preparation time compared to those usually involved by other conventional extraction techniques. The system can be easily coupled to different types of chromatographic purification and / or separation systems and with certain detectors.

State of the art

Pressurized liquid extraction (PLE) is a newly introduced sample preparation technique

(B.E. Richter, J.L. Ezzell, D. Felix, K.A. Roberts, D.W. Later, Am. Lab. 27 (1995) 24). However, this technique has undergone a great development in recent years because it represents a simple, fast alternative that implies a lower consumption of solvents and adsorbents than the conventional techniques used for the extraction of non-volatile compounds or of moderate volatility of semi-samples. -Solid and solid. The different fields of application and the different analytical approaches tested to date have been summarized and discussed in different reviews, such as J.L. Luque-García, M.D. Luque de Castro, Trends Anal. Chem. 23 (2004); 102-108., L. Ramos, E.M. Kristenson, U.A.Th. Brinkman J. Chromatography A, 975 (2002) 3-29.

To date, three PLE systems have been commercialized. The first has been commercialized since 1995 (US Patents 5,843,311, US 5, 647,976, US 5,660,727, US 5,785,856). There are two models of this PLE. The first reaches temperatures of up to 200 ° C and pressures of up to 21 MPa in extraction cells of 1, 5, 11, 22 or 33 ml. It allows the consecutive analysis of up to 24 samples whose extracts are collected in 26 vials of 40 or 60 ml. Recently, a variant has been introduced, with similar characteristics, but for the analysis of a single sample (the cell can be 10, 34, 66 or 100 ml and the vial for the extract is 250 ml). The second model is for larger samples, reaches similar temperatures but lower pressures (up to 10 MPa) in cells of 34, 66 or 100 ml, and allows the consecutive processing of up to 12 samples by collecting the extracts in 250 ml vials.

The number of PLE systems available in the market is limited and none of them is suitable for the treatment of small samples. Due to its characteristics and lack of flexibility of its designs, neither allows it to be connected online or with another cell for the consecutive treatment of the sample or with a chromatographic purification and / or separation-detection system. They also do not allow working with cells of different sizes simultaneously or sequentially, as does the new device to which this descriptive memory refers.

The first prior example of PLE miniaturized system was developed by one of the inventors of this patent (L. Ramos, J.J. Vreuls, U.A.Th. Brinkman. J. Chromatography A, 891 (2000) 275-286). This first miniaturized PLE system has been successfully used for the extraction of traces of environmental microcontaminants, such as polycyclic aromatic hydrocarbons (L. Ramos, JJ Vreuls, UATh. Brinkman. J. Chromatography A, 891 (2000) 275-286) and chloroanilines (EM Kristenson, S. Angioi, RJJ Vreuls, MC Gennaro, UATh. Brinkman, J. Chromatogr. A, 2004), of soils and sediments. The system allows the reproducible extraction of the endogenous analytes of interest with minimum volumes of organic solvents (about 100 µl) and with significant time savings compared to conventional sample treatment procedures (about 20 min vs. several hours). Its use combined with a sensitive and selective instrumental analysis system allows the unequivocal determination of the compounds studied without additional treatment of the extracts, which, combined with the reduced volume of solvent used, makes direct coupling between the PLE system and the separation-detection chosen. However, by design, the size of the cell to be used is limited to 10 mm × 9 mm outside diameter (o.d.), the internal diameter being the only parameter that can be modified (in the range 2-4 mm). The heating is carried out with a resistance wound around a metal ring inside which the cell is placed and the insulation with a ceramic ring surrounding the resistance.

Recently a miniaturized PLE system has been described, included in the Spanish patent ES2276592, which consists of a thermostated furnace of reduced dimensions in which several extraction cells can be mounted whose ends are connected to two valves equipped with a coil as a system of cooling. The cells consist of stainless steel tubes of variable size (in principle, 10-150 mm in length; 2-11 mm of ID; and 4.6-15 mm of ID). A liquid chromatography (LC) pump allows the solvent to be pumped and pressurized into the cell and the respective injection valves (six-way), connected to the inlet and outlet of the extraction cell. The device allows precise control of the volume of extractant and pressure applied to each cell, which allows the use of small cells and the extraction of 20-50 mg samples with minimal solvent and reagent costs.

This system has an important limitation related to the thermal inertia of the aluminum plate used to heat, due to the high time required for the system to reach the maximum working temperature (ca. 20 min) and to achieve complete cooling from the extraction cell and the sample after the extraction process (ca. 15 min).

Description of the invention

The designed PLE equipment incorporates a circular mounting resistor with a maximum power of 1800 W, inserted into a steel tube, into the system described in the original device (ES2276592). Behind is a motor that guides an adjustable air flow rate between ca. 250-500 L / min through the tube for heating the chamber where the extraction cells are placed. Both the resistors and the motor can be turned on and off independently. This device replaces that of three 140 W cartridge-type electrical resistors, inserted in the aluminum plate, which was used for heating in the original system. The camera has a “J” type thermocouple inserted for constant monitoring and regulation of the temperature through a digital electronic controller that works with a PID algorithm (Osaka OK48 / 482). The set is in an oven isolated from the rest of the system components and included in a 53 × 35 × 18 cm metal box on whose front the controller and valves are inserted (Figure 1), resulting in a compact device.

With this setting, the temperature of the extraction cell can be adjusted between 25 and 200ºC and reduce the time required by the system to reach the maximum working temperature. For cooling the device, the resistors are switched off while the engine is running, so that the initial operating temperature of the device (normally the ambient temperature) is reached in a much faster way, thus reducing the time needed to operate between two samples in a cell.

Otherwise, the system consists of a series of extraction cells inserted in an oven equipped with a temperature control and regulation system, a liquid chromatography (LC) pump to pump and pressurize the solvent in the cell and the respective valves injection (six-way), connected to the inlet and outlet of the extraction cell. While the high temperature accelerates the kinetics of the extraction process, the high pressure keeps the solvent in a liquid state at the high temperatures used and favors its penetration into the matrix, resulting in an efficient, fast and low consumption method of extraction. of solvents, which allows to obtain more concentrated extracts and occasionally ready to be analyzed. As it is a closed system, there is a lower risk of contamination and / or loss of analytes, the analyst's exposure to organic solvents is reduced and, due to the lower solvent consumption, it is more environmentally friendly than other systems. conventional treatments All instrumental parameters are controlled through a front panel, except for the pressure that is controlled with the LC pump. The system can be used independently or coupled in line with a graphical chromate purification and / or separation-detection system.

However, an external LC pump is required for pumping and pressurization of the system. The use of gases for the displacement of solvents is expendable, and the insertion of a coil of small internal diameter (id) at the outlet of the cell outlet valve allows rapid cooling of the extract without causing, in the case of Small samples, distortion of the transferred band, that is, without further dilution of the extracts. The maximum working pressure is 20 MPa and can be controlled independently for each extraction cycle in each of the cells.

The equipment allows the installation of up to three cells in the oven simultaneously, for independent extraction, and each of them can have different dimensions. The cells consist of stainless steel tubes of variable size (in principle, 10-150 mm in length; 2-11 mm of ID; and 4.6-15 mm of ID), although it is advisable that your ID. increase proportionally by doing so the length of the cell. At the ends of the cells, some material (paper fi lters, glass wool or, preferably, 0.5 μm metal chips) is arranged to prevent particles of the matrix and / or adsorbents from leaving it, which would cause blockage of the pipes and valves of the system. The fi lters (or fritters) are manually fixed using stainless steel screws that, in turn, allow the connection of Rheodyne nuts and LC steel tubes used to connect the ends of the cell with six-way Rheodyne valves. These valves allow the pressurization of the extractant pumped with an LC pump inside the cell. Once the extraction is complete, the extract can be (i) collected in a vial of adequate size, (ii) transferred directly, thanks to the valve system, to a chromatographic system suitable for purification and / or separation-detection in line, or (iii) transferred to the next extraction cell where it is subjected to additional treatment.

A detailed design of the PLE device in its different stages of operation is shown in Figure 1.

Description of the fi gures

The present invention is illustrated by the following figure 1, where a diagram of the Extractor with pressurized liquids is represented. A cell connected to two valves is shown in the pressurization stage and the different actuation possibilities of the valves.

The system consists of the following elements:

one.

Lower valve connections in “load” (α) and “inject” (β) position;

2.

Upper valve connections in “load” (α) and “inject” (β) position;

3.

Liquid chromatography (LC) pump;

Four.

Stainless steel extraction cell;

5.

Details of the extraction cell; to. fried b. splint, c. sample and dispersant mixture, d. Glass wool cap, e. lock nut with connection for LC tubes;

6.

Cooling coil;

7.

Collection of extract;

8.

Thermocouple type "J";

9.

Circular mounting resistor with a maximum power of 1800 W, inserted in a steel tube, coupled to the motor that guides an adjustable air flow between ca. 250-500 L / min through the tube for heating the chamber where the extraction cells are placed;

10.

Temperature controller;

eleven.

Interior of the oven.

Embodiment of the invention

By way of illustration of the aforementioned characteristics, and without this example being limiting with respect to the possibilities of application of the miniaturized extraction system with pressurized liquids referred to in the present specification, its application for extraction and purification is described fast, precise and in a single step, of 22 individual endogenous congeners of polychlorinated biphenyls (PCBs), including the less abundant non-and monoorto-substituted toxic isomers and indicators, present in fatty foods such as pork, chicken and fish (fat content, 49%, 22% and 25%, respectively).

Once all the aforementioned variables have been optimized and that may affect the efficiency of the extraction process and, in this particular application, the efficiency of the process of purification of the extracts by performing it simultaneously within the same extraction cell , the method develops the following steps:

-

A representative portion of the lyophilized food, ie about 500 mg, is weighed and mixed and homogenized in a mortar with similar amounts of anhydrous sodium sulfate and silica modified with sulfuric acid.

-

Then, 300 mg of this mixture, or corresponding to 100 mg of sample, is packaged in a 10 mm × 4.6 mm extraction cell. i.d. × 6.6 mm o.d. in whose lower part a metal frit was previously installed manually that was adjusted by a nut of suitable size. On the sample, 900 mg of silica modified with sulfuric acid and, on this, 60 mg of neutral silica are also packed in the cell until approximately 98% of the internal volume of the cell is completed. The frit is adjusted manually to the top of the cell and it is closed with the corresponding nut.

-

Once the LC tubes are adjusted to the ends of the cell, it is installed manually in the oven and the free ends of the LC tubes are adjusted manually in the corresponding ports of six-way valves.

-

Next, the temperature program is started on the PLE device designed with a resistance of 1800 W, which allows to reach the required temperature very quickly. Once the temperature finally chosen to carry out the extraction is reached, that is, from room temperature to 40 ° C, the solvent chosen as an extractant, n-hexane, is started to be pumped at 0.4 ml / min with the LC pump until it start to elute at the end of the capillary located in the valve (2). At that time, the valve is switched

(2) to position β and pressurization of the system up to 12 MPa finally set as the optimum working pressure in this method is started. When this pressure is reached, the valve (1) is switched to the β position and the actual extraction stage is started.

-

The method consists of two consecutive static extraction-purification cycles of 7 min each. Once the first static extraction cycle is completed, the valve (2) is switched and the extract is collected with the analytes in a 10 ml graduated vial through the coil installed in this same valve (2). Once the system has been depressurized, which happens instantaneously, the valve (1) is switched to the α position and 1.5 ml of n-hexane was pumped to displace the solvent used in the first extraction cycle of the cell and tubes. LC. When 1.5 ml of extract is collected in the vial, the process described in the previous point is repeated, except for the temperature programming, which remains constant throughout the entire experiment. Once the second 7-minute cycle has concluded, the procedure is similar to that described for the first cycle. The final volume of extractant collected is 3.5 ml and the PLE process is completed in 20 min. Once the extraction process has been completed and the PLE system is depressurized, the heating resistors are switched off, keeping the air motor on, so that the extraction cells and the coils are cooled in a time much shorter than the one required by the original device

-

Once the extraction is finished, manually, the valves (2) and (1) are switched back to their positions β, the LC tubes are disconnected from the valves, the oven cell is disassembled, the end screws are disconnected and empty the cell for cleaning and later use.

-

The extractant collected in the vial is concentrated and transferred to a 1 ml vial, where its final volume is adjusted to 10 μl, the internal standard is added, and analyzed without further treatment by GC-μECD (or GC-ITD ( MS / MS) in the case of less abundant non-ortho-substituted PCBs, ie at pg / g fat levels).

The present method allows a faster determination of the analytes investigated in complex samples of fatty foods with a minimum consumption of sample, solvents and time, allowing a complete sample preparation, that is, performing the extraction and purification in a single step within a closed and miniaturized system.

Despite the small sample size used, 100 mg, the method allows efficient and accurate extraction of analytes. The average concentrations of PCBs determined using the designed PLE system are 98% and 108% (with extreme values in the range 83-130%) of those obtained by using conventional extraction procedures, such as Soxhlet or matrix dispersion in solid phase, followed by the usual off-line purification processes for this type of study. The repeatability of the method was better than 23% in all cases and is considered a valid value for this type of analysis (levels in the ng-pg / g fat range); the linearity is greater than 0.94 in the investigated range of 1-10 ng / g sample, and the detection limits are between 0.009-0.04 ng / g sample for most of the compounds studied although only 1 μl of the extract is injected final in the GC-μECD system.

Claims (13)

1. PLE miniaturized device for extraction with liquids in closed cell and at high temperatures and pressures of semi-solid and solid samples, consisting of at least one extraction cell, an oven equipped with temperature regulation system, a chromatography pump liquid and injection valves connected to the inlet and outlet of the extraction cells, characterized in that the temperature regulation system comprises:

to.

at least one circular mounting resistor with a maximum power of 1800 W, inserted into a steel tube;

b.

at least one motor that guides an air flow through said tube;

C.

at least one control element that allows said resistance and said motor to be switched on and off independently.

2.

Miniaturized PLE device for extraction with liquids in closed cell and at high temperatures and pressures according to claim 1, characterized in that the resistance power is adjustable up to a maximum value of 1800 W.

3.

Miniaturized PLE device for extraction with liquids in closed cell and at high temperatures and pressures according to any of the preceding claims, characterized in that the engine that guides the air flow has a flow capacity between 250 and 500 L / min.

Four.

Miniaturized PLE device for extraction with liquids in closed cell and at high temperatures and pressures according to any of the preceding claims, characterized in that it incorporates at least one coil for cooling the sample.

5.

Miniaturized PLE device for extraction with liquids in closed cell and at elevated temperatures and pressures according to any of the preceding claims, characterized in that the injection valves are six-way.

6.

Miniaturized PLE device for extraction with liquids in closed cell and at elevated temperatures and pressures according to any of the preceding claims, characterized in that the volume of the extraction cells is between 0.05 and 30 ml.

7.

Miniaturized PLE device for extraction with liquids in closed cell and at high temperatures and pressures according to any of the preceding claims, characterized in that the dimensions of the extraction cells are between 10 and 150 mm in length, 2 and 11 mm in internal diameter and 4.6 and 15 mm external diameter

8.

Miniaturized PLE device for extraction with liquids in closed cell and at elevated temperatures and pressures according to any of the preceding claims, characterized in that the extraction cells can be coupled in line.

9.

Miniaturized PLE device for extraction with liquids in closed cell and at elevated temperatures and pressures according to any of the preceding claims, characterized in that the working pressure range in each extraction cell is from 1 to 20MPa.

10.

Miniaturized PLE device for extraction with liquids in closed cell and high temperatures and pressures according to any of the preceding claims, characterized in that the working temperature range is 25 to 200 ° C.

eleven.

Miniaturized PLE device for extraction with liquids in closed cell and high temperatures and pressures according to any of the preceding claims, characterized in that it can be coupled to a chromatographic analyte separation system.

12.

Miniaturized PLE device for extraction with liquids in closed cell and high temperatures and pressures according to any of the preceding claims, characterized in that it can be coupled to an analyte detection system.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201000541 SPAIN Date of submission of the application: 04.28.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: B01D11 / 02 (2006.01) G01N33 / 00 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

EN 2276592 A1 (UNIV MADRID COMPLUTENSE; SUPERIOR RESEARCH COUNCIL) 16.06.2007, figure 1. 1-12

TO

RAMOS, L. et al. "Miniaturized pressurised liquid extraction of polycyclic aromatic hydrocarbons from soil and sediment with subsequent large-volume injection-gas chromatography" Journal of Chromatography, September 8, 2000, Vol. 891, No. 2, pages 275-286; instrumentation and procedure, figure 1. 1-12

TO

KRISTENSON, E. M. "Miniaturized pressurized liquid extraction of chloroanilines from soil with subsequent analysis by large-volume injection-gas chromatography-mass spectrometry" Journal of Chromatography, November 26, 2004, Vol. 1058, No. 1-2, pages 243-249; Instrumentation and procedure. 1-12

TO

US 2003096422 A1 (NGO ENG SHI et al.) 22.05.2003, paragraph [44]. 1-12

TO

RAMOS, J.J. et al. "Miniaturized selective pressurized liquid extraction of polychlorinated biphenyls from foodstuffs" Journal of Chromatography, May 16, 2007, Vol. 1152, No. 1-2, pages 254-261; Instrumentation and procedure. 1-12

TO

WO 2004034021 A2 (FLUID MAN SYSTEMS INC) 04.24.2004, pages 6-7; figure 2. 1-12

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 01.08.2011

Examiner M. García González Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201000541 Minimum documentation sought (classification system followed by classification symbols) B01D, G01N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, TXT, XPESP, NPL State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201000541 Date of Completion of Written Opinion: 01.08.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-12 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-12 Claims IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201000541 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

EN 2276592 A1 (UNIV MADRID COMPLUTENSE; SUPERIOR COUNCIL RESEARCH) 06/16/2007

D02

RAMOS, L. et al. "Miniaturized pressurised liquid extraction of polycyclic aromatic hydrocarbons from soil and sediment with subsequent large-volume injection-gas chromatography" Journal of Chromatography, September 8, 2000, Vol. 891, No. 2, pages 275-286; instrumentation and procedure, figure 1. 08.09.2000

D03

KRISTENSON, E. M. et al. "Miniaturized pressurised liquid extraction of chloroanilines from soil with subsequent analysis by large-volume injection-gas chromatography-mass spectrometry" Journal of Chromatography, November 26, 2004, Vol. 1058, No. 1-2, pages 243-249; Instrumentation and procedure. 11.26.2004

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention is a miniaturized device for the extraction with pressurized liquids of semi-solid and solid samples. Document D01 discloses a miniaturized device for the extraction with pressurized liquids of semi-solid and solid samples consisting of several extraction cells (4), an oven (11) with temperature regulation system (10), a pump of liquid chromatography (3) and injection valves (1,2) at the inlet and outlet of the extraction cells, in which the heating is carried out by means of an aluminum plate (8) in which three resistors have been inserted electric cartridge type 140 W. (see references in figure 1) Document D02 discloses a miniaturized extraction process of polycyclic aromatic hydrocarbons with pressurized liquids in which the heating is carried out with a resistance wound around a metal ring in which the extraction cell is located. (see instrumentation and procedure and figure 1) Document D03 discloses a miniaturized extraction process of chloroanilines with pressurized liquids where the cell is surrounded with a steel ring connected to a resistance to heat it. (see instrumentation and procedure) None of the aforementioned documents or any relevant combination thereof discloses a miniaturized device for extraction with pressurized liquids in which the temperature regulation system comprises a circular mounting resistance inserted in a steel tube, a motor that guides a air flow through said tube and a control element that allows the resistance and motor to be switched on and off independently as set out in claim 1 of the application, with the associated advantage of reducing the time required for the system to arrive at the working temperature, as well as the cooling time until the initial operating temperature of the device is reached, so that the time needed to operate between two samples in a cell decreases compared to the usual techniques in the state of the art . Accordingly, the invention as set forth in claims 1-12 of the application is new and involves inventive activity. (Art. 6 and 8 LP) State of the Art Report Page 4/4