WO2024194405A1

WO2024194405A1 - Method and apparatus for the selective isolation of single or multiple compounds from complex mixtures

Info

Publication number: WO2024194405A1
Application number: PCT/EP2024/057577
Authority: WO
Inventors: Fabio Di Francesco; Matyas RIPSZAM; Tobias Andreas Christian BRUDERER; Federico Maria VIVALDI
Original assignee: Universita di Pisa
Current assignee: Universita di Pisa
Priority date: 2023-03-23
Filing date: 2024-03-21
Publication date: 2024-09-26
Anticipated expiration: 2025-09-23

Abstract

The present invention relates to a method and an apparatus for the identifying and isolating analytes from a sample comprising a complex mixture.

Description

METHOD AND APPARATUS FOR THE SELECTIVE ISOLATION OF SINGLE OR MULTIPLE COMPOUNDS FROM COMPLEX MIXTURES

Field of the invention

Present invention relates to an apparatus and a method employing it for the selective isolation of a single or multiple compounds from complex mixtures.

Background of the invention

Traditional one-dimensional Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition, by injecting a gaseous or liquid sample into a mobile phase, typically called the carrier gas, and passing the gas through a stationary phase. The mobile phase is usually an inert gas or an unreactive gas such as helium, argon, nitrogen, or hydrogen. The stationary phase is a microscopic layer of viscous liquid on a surface of solid particles on an inert solid support inside a piece of glass or metal tubing called a column. The glass or metal column, through which the gas phase passes, is located in an oven where the temperature of the gas can be controlled and the eluent coming off the column is monitored by a computerized detector. This technique had the drawback that the identification capability of the method is limited to the chemicals that are fully separated which posed difficulties when analyzing very complex samples. In order to solve these problems, comprehensive two- dimensional gas chromatography (GC*GC) has been developed, wherein the chemicals in the sample are first separated using a single dimension chromatographical system. At the same time, this chromatogram is being sliced (or modulated) into few second long subdivisions using a purge-and-trap device where they are also separated using an additional, short chromatographic column with a different selectivity. Using this technique, the initial separation achieved by the first column is retained, whilst performing an additional dimension of separation. This allows to achieve a much higher spectral purity and better quality of library searches for chemical identification, due to the lack of interferences considering the powerful separation capabilities. Also, the number of detected peaks is usually at least 10-fold higher in GC*GC, while analyzing the same sample, in comparison to single dimension GC. Furthermore, chromatographic peak shapes are sharp, i.e. peak widths of 50 - 200 ms instead of 5-8 s in single dimension. This will be important because of the switching speed. Hence, advantages of GC*GC, and specifically of flow-modulated GC*GC, include increased peak capacity, improved resolution, and unique selectivity and sensitivity enhancement compared to conventional one-dimensional gas chromatography (ID GC). By coupling GC with a mass- selective detector (such as a single quadrupole or a quadrupole-time-of-flight mass spectrometer Q-TOF) instead of a non-selective flame ionization detector (FID), it is also possible to further increase the potential of the analytical technique, in order to maximize the output. Furthermore, with the introduction of microfluidic switching devices, it is possible to isolate selected peaks from the sample mixture. However, when using a switching device in a GCxGC system, very fast switching is required to be able to isolate single components because of the very narrow chromatographic peaks.

Nevertheless, a first problem with known devices and known methods is the change in viscosity of gases (as a function of temperature) through long capillaries (up to 80 cm) and the consequent need of providing high pressures/flows to them, the lack of which would contribute enough to hamper the switching. In fact, the laminar flow rates are inversely proportional to the gas temperature. Since there is a linear temperature program in the GC oven, the pressure at the column head should be increased according to the temperature to maintain the constant gas flow in the capillary columns. If one segment of the column is kept at a constant temperature and the other segment follows a dynamic temperature program, it causes the flow characteristics to drastically change during the GC run which would make the fast and efficient switching impossible for the isolation of single chemicals.

In known devices, this problem is dealt with via the introduction of a cold trap inside the GC oven, that is connected to a heated transfer line that is usually kept at a constant temperature (e.g., 200 °C), where isolated peaks are collected and afterwards, desorbed through a heated transfer line.

This solution has the disadvantage that a further device, namely the cold trap, should be installed and carefully programmed inside the GC oven, specifically using a large amount of liquid CO2 or liquid N2, to precisely control the temperature.

Moreover, a second problem which is still not solved, is that complex mixtures, such as chemical mixtures coming from food and fragrances samples are still very difficult to analyze and more importantly it is still very difficult to isolate single and distinct peaks of a chromatogram even with GCxGC, to identify the analytes represented by these peaks and subsequently separate said analytes from the starting complex mixtures.

In fact, there is still the need of a new approach that allows very complex mixtures to be separated and selectively analyzed as single or multiple compounds in order to be detected.

Summary of the invention

The present invention solves the above-mentioned problem by providing a GCxGC apparatus for the selective isolation of a single or multiple compounds from complex mixtures comprising:

- a heated inlet (1) for the injection of a liquid, gas, or solid sample and of a gas carrier,

- a first GC column (2) and a second GC column (3) placed in a GC oven (13) with interposition of a flow modulator (4) in between;

- a Deans switch valve (5) placed downstream with respect to the first (2) and the second (3) column and having:

- a first outlet (5a) connected through a capillary (6) to a passive splitter (7), apt to divide the flow of the vaporized liquid, gas or solid sample in a gas carrier into at least two lines, preferably two lines, a first transfer line comprising a transfer capillary (7a), directed to a Q-TOF-FID-MS detector (10), and a second isolation line comprising an isolation capillary (7b), directed to a recovery capillary (7c) for the isolation of the sample;

- a second outlet (5b) connected through a capillary (8) with a waste line (9); and wherein each of the capillaries (6, 7a, 7b and 8) dimensions are calculated through the equation: where:

Ap is the pressure difference between the two ends of the capillary (6, 7a, 7b and 8),

L is the length of the capillary (6, 7a, 7b and 8), p is the dynamic viscosity of the gas carrier, Q is the volumetric flow rate of the gas carrier, R is the capillary (6, 7a, 7b and 8) radius; and wherein the capillary (7c) is at least partially refrigerated via a cooling device (11).

The invention further relates to method for identifying and/or separating analytes from a sample, comprising the steps of: a) providing a liquid, gas or solid sample to be analyzed; b) inserting the sample and a gas carrier into the heated inlet (1) of the apparatus of the invention, thus obtaining a vaporized sample; c) conveying the vaporized sample with a flow of carrier gas through a first column (2) and a second column (3), with interposition of a flow modulator (4) in between, such that the second flow rate for the second column (3) is higher than the first flow rate of the first column (2); d) conveying the sample from column (3) through a valve (5) into two alternative flow lines: an isolation line dl), wherein the sample flows from the valve (5) to the first outlet (5a), while the second outlet (5b) is closed, via a capillary (6), thus reaching, through a passive splitter (7), the Q-TOF-MS (10) through the transfer capillary (7a) and the recovery capillary (7c) through the isolation capillary (7b); or a divert line d2), wherein the sample flows from the valve (5) to the second outlet (5b), while the first outlet (5a) is closed, via a capillary (8), thus reaching the waste line (9); wherein the valve (5) is operated to switch from isolation line (dl) to the divert line (d2); e) identifying and/or separating the analytes flowing from the isolation line dl).

All the novel modifications allow a range of new applications, e.g., the isolation of single compounds from food/fragrance samples, the investigation of the toxicological effects through isolation of individual or multiple known or unknown suspect chemicals, effect studies in biomedical and pharmaceutical fields in model organisms (mice) and on humans, such as cell line testing, bacteria cultures, human enzyme testing (P450 activation) and the isolation and separation from the complex mixture of single compounds for further chemical characterization for structure elucidation. Hence the method of invention is for the isolation and separation from a complex mixture of single analytes, wherein the complex mixture is preferably a food or fragrances.

Brief description of Figures

Figures 1A-B: show the schematic of the GC*GC apparatus setup of the invention. Segment 1 in Fig. lA is the chromatographic separation part, Segment 2 in Fig.lA is the microfluidic switching part and Segment 3 in Fig.1 A is the isolate collection part.

Figures 2A-B and Figures 3A-B show the two different states of the Deans switch of the apparatus of the invention.

Figure 4A shows the problem, within the transfer line, of the gas viscosity in relation to the temperature change inside long capillaries; and Figure 4B shows the solution of the present invention, where the apparatus of the invention used an adapter (12) for the FID port of the MS detector (10).

Figure 5a.) shows the schematic of the FID port on an Agilent GC, where the screws (1) and the jet (2) need to be removed. The part that needs further modification to install our modification is selected and magnified. Figure 5b.) show the parts that need to be removed to have the FID collector (3) free and Figure 5c.) shows the structure in detail of the adapter (12) mounted on the FID outlet, where (11) is the Peltier cooling element having dimensions of 6cm * 4cm * 1cm.

Figures 6A-C show respectively, the overlaid unprocessed two-dimensional total-ion chromatograms (TIC) of the original sample (Fig.6A), the residual/subtractive chromatogram (Fig.6B) and the isolated chromatogram (Fig.6C)_and according to Example 2. The x-axis is the overall retention time, the y-axis is the relative intensity (to the highest peak) displayed in a linked axis mode.

Figures 7A-B show the residual/subtractive total ion chromatogram (TIC) according to Example 2. In the zoomed image (Figure 7B) it is shown the time for the baseline stabilization.

Figures 8A-C show the total ion current (TIC) according to Example 2, where the chemicals selected to be isolated are signaled by white rectangles with solid line and arrows with their name. Retention time I represents the retention of chemicals on the first-dimension column expressed in minutes, retention time II represents the degree of retention on the second column, expressed in seconds. Fig. 8A and Fig 8A’ show a traditional GC*GC chromatogram for a complex mixture, Fig. 8B and Fig. 8B’ show the subtractive isolation GC*GC chromatographic runs and Fig. 8C and Fig 8C’ show only the selectively isolated chemicals from the standard mixture.

Figure 9A-B respectively represent the zoomed contour plots based on total ion current of the standard mixture according to Example 2 (Fig.9A) and the selectively isolated (Fig.9B) peak of Methyl eugenol.

Detailed description of the invention

The inventors surprisingly found a new approach to identify and/or separate single compounds or analytes from complex mixtures which have been analyzed by comprehensive GC*GC chromatography and still are either not chemically known or have co-eluting peaks that are difficult to be isolated and thus correctly analyzed in order to recognize the chemical nature of the single analyte.

Through the apparatus of the invention and a method employing it, they were able to selectively isolate peaks that are baseline-separated in a 2D plane with a minimum distance of 50 milliseconds from other eluting peaks. They were further able to selectively subtract unwanted peaks, and selectively separate from the complex mixture a sample comprising the separated/recovered analyte to be further analyzed.

This was made possible by modifying a GC*GC system with a Deans switch valve (5), a passive splitter (7), careful adjustments of specific flow and pressure ranges through the capillaries of the apparatus of the invention and via specific steps of a method for identifying and/or separating analytes comprised in a sample employing said apparatus of the invention.

The apparatus of the invention and the method employing it was tested, as will be clear from the experimental part, with a large number of reference compounds, covering a wide range of volatility and polarities, from low boiling point compounds, such as acetone (having boiling point at 55.6°C) to high boiling points compounds, such as heptadecane (having boiling point at 343°C), passing through several middle boiling point compounds, such as 2-pentanol (having boiling point at 119.4°C) and 2-methyl-l -Propanol, 1-Butanol, 2-Butanone, 3-methyl-2- Pentanone, 3 -Pentanone, 2-Pentanol, 3 -Pentanol, Eucalyptol, Benzene acetaldehyde, etc. Among all the compounds tested, co-eluting compounds, having peaks at least partially overlapped, have been chosen and further tested, namely 2,6-dimethyl-4-heptanone (CAS: 108- 83-8), 3-methyl cyclohexanone (CAS: 591-24-2), Methyleugenol (CAS: 93-15-2) and Irans- - Damascenone (CAS: 23726-93-4), 1-butanolo, 2-butanolo, 2-pentanol, 3 -pentanol, 2- pentanone, 3-pentanone, 1 -methyl- 1 -butanol, methyl isobutyl ketone, in order to show the advantages of the present apparatus and method employing it, as will be shown and demonstrated in the experimental part.

Moreover, the isolation and recollection from tube to tube till reaching the recovery capillary (7c) is highly efficient with the use in the system of the invention of an adapter (12), which allowed higher recovery values and ensured uniformity of temperatures and viscosity of the gases employed, throughout the apparatus and the method. Existing software could be used to program the isolation or removal of features respective compounds of interest.

In the present invention, with the following definitions:

“molecules” and “compounds” it is meant any compound or analyte present in a complex mixture and can be used interchangeably;

“GC*GC” it is meant Comprehensive Two-dimensional Gas Chromatography, and it is a multidimensional gas chromatography technique which utilizes two columns with two stationary phases of different selectivity. In GC*GC, all the effluent from the first-dimension conventional column (30 - 60 m) is transferred to the second dimension a short-fast column (0.5 - 5 m) via a modulator, which traps, then "injects" the effluent from the first-dimension column onto the second dimension, hence creating a retention plane of the 1^st dimension separation x 2^nd dimension separation.

“Q-TOF” or “Q-TOF-MS” it is meant a ‘tandem’ instrument combining quadrupole technologies with a time-of-flight (TOF) mass analyzer (MS). Q-TOF-MS instrumentation closely resembles that of a triple-quadrupole mass spectrometer, though the third quadrupole has been replaced by a time-of-flight tube (TOF).

“FID” it is meant a flame ionization detector, where electrodes are placed adjacent to a flame fueled by hydrogen/air near the exit of the column, and when carbon containing compounds exit the column, they are pyrolyzed by the flame. FID compatible carrier gasses include helium, hydrogen, nitrogen, and argon.

“GCxGC-Q-TOF-MS-FID” it is meant a two-dimensional gas chromatography coupled with both a Q-TOF-MS detector, which is highly effective and sensitive, even in a small quantity of sample, and an FID detector or FID port;

“Deans switch” it is meant a microfluidic plate that is placed inside the GC oven. It has one inlet and two identical outlets. The outlets are connected to a solenoid valve that facilitates the auxiliary carrier gas pressure and the switching. The state of this valve and the dimensions of the capillaries which are attached to the Deans switch determine the direction of the flow. The carrier gas is supplied by an auxiliary EPC unit which is connected to the solenoid valve;

“EPC” it is meant an electronic pressure control, which controls the pressure in the Deans switch;

“approximately” it is meant ± 5 s;

“psi” it is meant pounds per square inch, equal to 6894.76 Pa or 0.0689 bar;

“co-eluting compounds” it is meant chemical substances, which normally cannot be separated using a chromatographic system, since their retention times are too close to be resolved by the GC at the current method settings;

“deactivated silica” it is meant that the reactive sites (silanol groups) on the surface of the silica capillary are deactivated using a reaction with trimethyl silyl chloride to prevent interaction between the chemicals and the free hydroxyl groups on the inner surface of the capillary.

In the present application, all values indicating the dimensions of tubes/capillaries/columns are expressed in length (m) * internal diameter (mm) * coating thickness (pm).

In a first aspect the invention relates to a GCxGC apparatus for the selective isolation of a single or multiple compounds from complex mixtures comprising:

- a first outlet (5a) connected through a capillary (6) to a passive splitter (7), apt to divide the flow of the vaporized liquid, gas or solid sample in a gas carrier into at least two lines, preferably two lines, a first transfer line comprising a transfer capillary (7a), directed to a Q- TOF-MS detector (10), and a second isolation line comprising an isolation capillary (7b), directed to a recovery capillary (7c) for the isolation of the sample;

- a second outlet (5b) connected through a capillary (8) with a waste line (9); and wherein each of the capillaries (6, 7a, 7b and 8) dimensions are calculated through the equation: Ap = — — where: 71 R

L is the length of the capillary (6, 7a, 7b and 8), p is the dynamic viscosity of the gas carrier, Q is the volumetric flow rate of the carrier gas, R is the capillary (6, 7a, 7b and 8) radius; and wherein the capillary (7c) is at least partially refrigerated via a cooling device (11). Advantageously, the inlet (1) temperature is comprised from 400 to 100°C, preferably from 300 to 200°C.

The sample injected via inlet (1) is a liquid sample, a gas sample or a solid sample, preferably a liquid sample.

In one embodiment, the liquid sample has a boiling point comprised in the range from 56 to 343°C.

In one embodiment, the liquid sample has a polarity comprised in the range from logP 0.2 to logP 9.4.

In one embodiment the gas carrier can be helium (He) and has a dynamic viscosity of 2 Pa *s at 30°C and 3.12 Pa*s at 300°C.

In another embodiment, the gas carrier can be nitrogen (N2) and has a dynamic viscosity of 1.8 Pa *s at 30°C and 2.86 Pa*s at 300°C.

In one embodiment the gas carrier has a volumetric flow rate comprised in the range of 0.5 - 1 mL/min.

Advantageously, the dynamic viscosity of the gas carrier p is defined according to the 5 k^mT i hard-sphere kinetic theory, hence calculated through the equation: p = 1.016 * ^^ ( )z where: p is the dynamic gas viscosity;

T is the gas temperature; c is the diameter of the gas particles; m is the mass of the gas particles; and kB is the Boltzmann constant, namely 1.380649 x 10 ²³ joule per kelvin (K), or 1.380649 x 10 ¹⁶ erg per kelvin.

Preferably, the sample injected via inlet (1) is a liquid sample, more preferably comprising at least two co-eluting compounds, hence having at least partially overlapped GC peaks.

In this embodiment the liquid sample is dissolved in a solvent, for example a non-polar solvent selected among any GC-compatible solvent known in the art, preferably selected from the group consisting of pentane, hexane, cyclohexane, isooctane, methyl-tert-butyl ether, dichloromethane, toluene, acetone.

The liquid sample is then injected into the inlet (1), which has preferably a temperature from 400 to 100°C, preferably from 300 to 200°C.

The temperature of the inlet (1) and the presence of a GC oven (13) where the first GC column (2) and the second GC column (3) are placed allow the vaporization of the solvent and volatile compounds in the sample, thus obtaining a vaporized sample. The presence of a flow of carrier gas into the inlet (1), allow the dispersion of the vaporized sample in the carrier gas. The carrier gas is also responsible for conveying the compounds to the columns, where the separation occurs.

In another embodiment, when the sample is a solid sample or a gas sample, the inlet (1) is a thermal desorber (1).

In the embodiment where the sample is a gas sample, the gas is injected into the thermal desorber (1), being a tube loaded with an absorbent, such as poly 2,6-diphenyl diphenylene oxide (Tenax), active carbon-based sorbent materials (Carbopack, Carbograph or Caxboxen), traps the gas sample on the surface of the absorbent. The thermal desorber (1), has preferably a temperature from 400 to 100°C, preferably from 300 to 200°C, and hence the trapped compounds are thermally desorbed before being conveyed, together with the carrier gas, to the columns, where the separation occurs.

In the embodiment where the sample is a solid sample, the solid is directly placed into the thermal desorber (1), which has preferably a temperature from 400 to 100°C, preferably from 300 to 200°C, and hence the volatile compounds desorb directly from the solid before being conveyed, together with the carrier gas, to the columns, where the separation occurs.

The first and the second columns (2 and 3) are GC columns, hence being placed in a GC oven (13).

Preferably, the first column (2) comprises a film of functionalized poly dimethyl siloxane having a thickness in the range from 0.1 to 1 pm, preferably from 0.25 to 1 pm, in order to increase the loadability and the retention times of the compounds contained in the sample.

Advantageously, the modulation period, hence each collection/re-inj ection cycle modulated by the flow modulator (4) from the first column (2) to the second column (3) was set in the range from 4 to 1 sec, preferably from 3 to 1.5 sec.

In a preferred embodiment, the first column (2) comprises a pneumatical controller module (PCM A) (F) apt to control the pressure of the sample and of the gas carrier, thus modulating the flow of the sample from the inlet (1) to the first column (2).

The gas carrier can be helium (He), nitrogen (N2) and mixtures thereof.

In the embodiment where the gas carrier is helium (He), the gas particles have, for example, an approximate diameter, based on the kinetic theory, of 260 pm, approximated as the median path length.

In the embodiment where the gas carrier is nitrogen (N2), the gas particles have, for example, an approximate diameter, based on the kinetic theory, of 364 pm, approximated as the median path length.

Advantageously, the PCM A (F) provides a pressure to the first column (2) which is calculated and adjusted automatically by the apparatus of the invention, based on the flow rates and the column dimensions (according to the equation of Hagen-Poiseuille) and the temperature and the gas carrier viscosity.

Preferably, the first column (2) has a constant flow of the liquid sample lower than the constant flow of the liquid sample of the second column (3), more preferably it is at least 10 times lower.

In a preferred embodiment, the first and the second column (2 and 3) has a constant flow of the liquid sample of, respectively and independently from one another, 0.5 mL*min'¹ for the first column (2) and 20 mL*min'¹ for the second column (3). These specific ranges allow a complete desorption of the first column (2).

The use of a flow-modulator (4) in between the two columns (2) and (3) is advantageous for the apparatus of the invention. The position of the flow modulator (4) between the two columns (2 and 3) has the function of continuously collecting small fractions of the effluent from the first column (2), ensuring that the separation is maintained in this dimension; focusing or refocusing the effluent of a narrow band; and quickly transferring the fraction collected into the second column (3) and focusing it as a narrow pulse. The basic operation principle of the flow modulator is collecting effluent fractions. Every collection/re-inj ection cycle, from the first column (2) to the second column (3), lasts approximately a few seconds, which is in accordance with the total length of the second-dimension separation segments, also considered as the length of the second-dimension chromatograms. The re-focusing of the chemicals is achieved by using high second column (3) flow rate of 20 mL/min'¹.

To supply this flow rate, a high pressure, a pressure valve (4’) is comprised in the flow modulator (4) and provides high pressures, from 30 psi at 40°C to 75 psi at 260°C throughout the whole method of the invention, at the head of the second column (3). This allows higher flows from the second column (2), comprised in the range above, to be provided also at the pressure control point (5’) of a valve (5).

The valve (5) is a Deans switch (5), more preferably it is a Deans switch (5) comprising an auxiliary electronic pressure controller (EPC) as pressure control point (5’), and a solenoid valve connected to the EPC. More preferably, the microfluidic plate of the Deans switch (5) is placed inside the GC oven (13) and the solenoid valve of the Deans switch (5) is fixed on top outside of the GC oven (13).

In a preferred embodiment, the valve (5) comprises pressure control point (5’) providing a pressure of the carrier gas in the range from 10 to 20 psi.

These specific pressures allow an almost instantaneous switch of the valve (5) from a first isolation line dl), wherein the liquid sample flows from the valve (5) to the first outlet (5a), while the second outlet (5b) is closed, via a capillary (6), to a second divert line d2), wherein the liquid sample flows from the valve (5) to the second outlet (5b), while the first outlet (5a) is closed, via a capillary (8).

Preferably, the capillary (6) is a deactivated silica capillary (6).

The pressure of the valve (5), in combination with the flow rate of the second column (3), allows to achieve a satisfactory separation of the final GC peaks of the complex sample mixture, whilst maintaining the narrowness of said peaks, hence maintaining the selectivity of the method. In fact, usual capillary GC flow rates (1-2 mL/min'¹) allow much slower stabilization, which would compromise the efficiency and the rate of switching, thus compromising the measurement. In order to isolate completely separated peaks, after each valve (5) switch, the flow needs to stabilize really fast. Using the apparatus of the invention, the flow stabilizes in approximately 50 ms (as also shown in Figure 7B), thus enabling a satisfactory separation of at least two co-eluting compounds, preferably having at least two peaks separated by 50ms of eluting time.

The passive splitter (7) is apt to divide the flow of the sample into at least two lines, preferably two lines: a first transfer line comprising a transfer capillary (7a), directed to a Q- TOF- MS detector (10), in order to identify the analytes by their mass spectrum, and a second isolation line comprising an isolation capillary (7b), directed to a recovery capillary (7c) for the isolation of the selected analytes from the sample.

Advantageously, in the first transfer line, the transfer capillary (7a) is an uncoated deactivated silica capillary.

Preferably, the second isolation line comprising the isolation capillary (7b), is directed into a recovery capillary (7c) for the separation of the sample, preferably being a sorbent tube containing an adsorbent, preferably poly 2,6-diphenylphenylene oxide.

Advantageously, in the second isolation line, the isolation capillary (7b) is an uncoated deactivated silica capillary.

Preferably, the capillary (8) is an uncoated deactivated silica capillary.

The capillaries (6, 7a, 7b and 8) dimensions are calculated with the Hagen-Poiseuille equation: where:

Ap is the pressure difference between the two ends of the capillary (6, 7a, 7b, 8 and 9) and (8),

L is the length of the capillary (6, 7a, 7b and 8), p is the dynamic viscosity of the gas carrier,

Q is the volumetric flow rate of the gas carrier,

R is the capillary (6, 7a, 7b and 8) radius.

Advantageously the ideal length L, calculated via the equation, was further adjusted by iterative practical optimization.

Preferably, the dimensions are calculated to match the flow characteristics of the capillaries connected to the two outlets (5a and 5b) of the valve (5) in order to establish an immediate and quantitative splitting of the flow of the sample. When the dimensions of the capillaries (6 and 8) connected respectively to the two outlets (5a and 5b) were significantly different, the second column effluent was flowing towards the gas line with the least amount of resistance.

Preferably, the capillary (6) has dimensions of 0.1m * 0.25mm.

Advantageously, the transfer capillary (7a) has dimensions of 3m * 0.18mm.

In a preferred and advantageous embodiment, the isolation capillary (7b) has dimensions of 0.8m * 0.25mm.

In a further preferred embodiment, the capillary (8) has dimensions of 0.7m * 0.25mm.

In a preferred and advantageous embodiment, the split ratio between the transfer line comprising the transfer capillary (7a) and the isolation line comprising the isolation capillary (7b) is comprised in the range from 11 :0.5 to 7:3, preferably 10: 0.7, more preferably is 9: 1, depending on the lengths/widths of the composite capillary (7a, 7b).

These specific split ratios allow to achieve the narrowest sample band, hence a more sensitive measurement of the analytes contained in the sample.

In a preferred embodiment, the above equation, namely the Hagen-Poiseuille equation, can be used to calculate and hence predict the flows, based on the capillaries (6, 7a, 7b and 8) dimensions and the pressure values can be adjusted accordingly at each pressure control point (T, 4 ’and 5’) in order to further match the temperature program.

The second outlet (5b) is connected through an uncoated deactivated silica capillary (8) with the waste line (9) and has an open end inside a GC oven (13).

Preferably, the temperature program set for the GC oven (13) is starting from a temperature in the range from 35 to 45°C, preferably is 40°C. Advantageously, the ramping temperature is 3°C/min. More preferably after reaching 4°C, the temperature programs hold for at least 30 seconds, even more preferably for 60 seconds.

After restarting the temperature program, with the ramping temperature in the range from 1 to 4°C/m, preferably is 3°C/min, the program is hold for a time comprised from 20 to 40 minutes, preferably from 20 to 30 minutes, more preferably for 21 minutes, till a temperature in the range from 200 to 260°C, preferably of 260°C is reached.

The capillary 7c of the apparatus of the invention is at least partially, preferably for at least 1cm of its length, refrigerated via a cooling device (11). The presence of the cooling device (11) allows to minimize breakthrough of the low-boiling chemicals in the complex mixture.

Preferably, the cooling device (11) is a Peltier cooler. More preferably, the cooling device (11) is a Peltier cooler, even more preferably having dimensions of 6cm * 4cm * 1cm and comprising an adapter (12), for holding the capillary (7b) attached to the FID port of GC oven (13).

The adapter (12) is preferably made of polycarbonate, to be more heat resistant and is compatible with all GC systems that have a flame ionization detector (FID) and is mounted using the screw holes. The adapter (12) may comprise a heat insulating layer, preferably a heat insulating adhesive, more preferably having a thickness of 3mm and being made of condensation repelling polyvinyl chloride (PVC), a circular septum made of poly dimethyl siloxane having a diameter of 11mm, and an O-ring having inner diameter of 5.92 mm and an outer diameter of 8mm to ensure fast and efficient sample transfer and a Peltier cooling element that will cool the adapter body to 0-5 °C, which will reduce the breakthrough of very volatile chemicals. An example of commercially available septum and O-ring is, respectively no. 5183- 4761 from Agilent and a ring produced by Markes. Using the adapter (12) with a FID shaft kept at high temperatures (e.g., at 300 - 400 °C) in the apparatus of the invention, instead of a long, heated capillary, it is possible to avoid the formation of cold spots that would lead to condensation and inefficient transfer of the analytes and, at the same time, minimize the contribution of the high-viscosity segment of the gas to a section of 1-2 centimeters. In fact, known commercially available heated capillary would be 80 cm and, if used the constant temperature of the heater would change the resistance of the gas line used for isolation. Performing the switching at low oven temperature (Figure 4a.), with the temperature set higher in the transfer line comprising a transfer capillary (7b), the relative resistance of the heated transfer line is increased. This will result in the switching being impossible because the resistance will be higher towards the isolation line comprising the isolation capillary (7b). On the contrary, if the switching is set up at high oven temperature (e.g., 250 °C) (Figure 4b.), the switching at lower temperatures will be hindered because of the relative decreased flow resistance in the isolation line comprising an isolation capillary (7b). Moreover, the isolation and recollection from tube to tube is highly efficient with the use in the apparatus of the invention comprising the specific adapter (12) attached to the FID port, since a recovery of 76 ± 7% can be achieved.

The Peltier cooler (11) comprising the adapter (12) is represented in Figures 5A-B).

By introducing this modification of the flame ionization detector (FID) port, the high viscosity gas contribution developed by heating the detector shaft to 300 °C is kept at a minimum (5 cm), and the formation of cold spots is avoided, while the adapter (12) facilitates the efficient collection of the selectively or subtractively isolated chemicals.

In a second aspect the invention relates to a method identifying and/or separating analytes comprised in a sample, comprising the steps of: a) providing a liquid, gas or solid sample to be analyzed; b) inserting the sample and a gas carrier into the heated inlet (1) of the apparatus of the invention, thus obtaining a vaporized sample; c) conveying the vaporised sample with a flow of gas carrier through a first column (2) and a second column (3), with interposition of a flow modulator (4) in between, such that the second flow rate for the second column (3) is higher than the first flow rate of the first column (2); d) conveying the sample from column (3) through a valve (5) into two alternative flow lines: an isolation line dl), wherein the sample flows from the valve (5) to the first outlet (5a), while the second outlet (5b) is closed, via a capillary (6), thus reaching, through a passive splitter (7), the Q-TOF -MS (10) through the transfer capillary (7a) and the recovery capillary (7c) though the isolation capillary (7b); or a divert line d2), wherein the sample flows from the valve (5) to the second outlet (5b), while the first outlet (5a) is closed, via a capillary (8), thus reaching the waste line (9); wherein the valve (5) is operated to switch from isolation line (dl) to the divert line (d2); e) identifying and/or separating the analytes flowing from the isolation line dl).

Advantageously, the insertion of step b) is a liquid injection or a thermal desorption, more preferably a liquid injection, even more preferably it is selected from a hot split liquid injection and a solvent venting liquid injection.

Advantageously, the hot split liquid injection results in better chromatographic peaks shape, in terms of intensity and sharpness.

If step b) is liquid injection, before step b) of providing a sample it is present a step b’) of dissolving an analyte or a mixture of analytes in a non-polar solvent selected among any GC- compatible solvent known in the art, preferably selected from the group consisting of pentane, hexane, cyclohexane, isooctane, methyl-tert-butyl ether, dichloromethane, toluene, acetone, thus obtaining a sample.

In a preferred embodiment, if step b) is liquid injection as above described, the sample of step b) is a liquid sample, preferably the liquid sample has a concentration in the range from 100 pg/pm to 100 ng/pm. The compounds are present in minimal quantities, so they do not contribute to altering the macroscopic properties of the carrier gas.

Advantageously the analytes comprised in the sample, comprise at least two co-eluting compounds, preferably the at least two co-eluting compounds are compounds having at least two peaks separated by 50ms of eluting time.

Alternatively, if step b) is a thermal desorption, the sample of step b) is a gas sample or a solid sample, and preferably step b) is a thermal desorption using as inlet (1) a thermal desorption device (1).

In this alternative embodiment, the sample of step b) is thermally desorbed as above described and preferably the thermally desorbed sample has a concentration in the range from 100 pg/pm to 100 ng/pm. The compounds are present in minimal quantities, so they do not contribute to altering the macroscopic properties of the carrier gas.

In both cases the sample of step b) has a volume comprised from 0,1 to 100 pL.

Preferably, step d) comprises two alternatives:

- a step dl’), where the valve (5) switches from the isolation line dl) to the divert line d2), wherein the sample flows from the second column (3) and is directed directly to the waste line (9), thus removing the analyte; or

- a step d2’), where the valve (5) switches from the divert line d2) to the isolation line dl), where the sample flows from the second column (3) to a passive splitter (7) which divides the flow towards:

- a capillary (7a) towards a Q-TOF -MS detector (10), thereby identifying the analyte; and

- a capillary (7b) towards a recovery capillary (7c), thereby isolating and recovering the analyte; wherein the analyte removed in step dl’) and the analyte isolated in step d2’) correspond to GC peaks which are thereby subtracted and isolated-identified, respectively.

Advantageously, step d) is achieved by programming the solenoid valve comprised in the valve (5) to act automatically via a software.

Any known software available on the market is suitable for this use, preferably MassHunter is used.

Preferably, in step d2’) the recovery in the recovery capillary (7c) of the separated sample is achieved in a sorbent tube attached to the capillary (7c) via a Swagelok joint with a septum, to ensure that the connection is airtight, and the sample recollection is complete.

With the method of the invention coupled with the apparatus of the invention, the isolation and recollection from tube to tube is highly efficient, since a recovery of 76 ± 7% can be achieved. All the other preferred conditions for the method of the invention are disclosed in detail with reference to the apparatus of the invention.

The method of the invention and the apparatus of the invention can be used for the isolation and separation from a complex mixture of single analytes, wherein the complex mixture is preferably a food or fragrances mixture.

The invention will be further supported by the following experimental part, which is intended as only exemplary and not limiting.

Experimental part

Materials and methods

The inlet (1) of the GC (Agilent 7890B - Agilent Technologies, Santa Clara, USA) was connected to the thermal desorber transfer line, thus acting as thermal desorber (1) from Markes TD. The comprehensive GC*GC instrumentation consisted of a dual column setup (first and second column 2 and 3) with a flow modulator (4) in between. The first column (2) was a J&W DB-5MS UI (30m * 0.25 mm * 1 pm) with a 1 pm film of modified poly dimethyl siloxane with 5% of diphenyl siloxane added. The second column was a J&W DB-WAX (5 m * 0.25 mm * 0.15 pm). Thermal desorption was performed using a Centri multi-purpose sampling platform. A flow of helium as gas carrier was supplied. The Deans switch (5) was connected to an uncoated silica capillary (6) with dimensions of 0.1 m * 0.25 mm, a passive splitter (7), a transfer capillary (7a) with dimensions of 3 m * 0.18 mm to the Q-TOF-FID-MS detector (10), and an capillary (7b) with dimensions of 0.8 m * 0.25 mm, and a deactivated silica capillary (8) with dimension of 0.7 m * 0.25 mm, with an open end inside the GC oven (13). The recovery tube (7c) was a sorbent tube containing poly 2,6-diphenylphenylene oxide, being TenaxGR produced by Merck and was cooled with a Peltier cooler (Supercool DA-020-12-02, Laird thermal systems) with an AT 12 stabilized power supply (K.E.R.T., Italy). The cooler was further modified with an adapter (12) as described above and as shown in Figures 5A-C. The O-ring used in the adapter (1) is produced by Markes International. Additionally, to minimize the temperature fluctuation, the aluminum section was covered with a heat insulating adhesive having a thickness of 3mm and being made of condensation repelling polyvinyl chloride (PVC) from Nova, Italy or 3M. The test mixture (3 pL) was spiked onto adsorbent tubes loaded with TenaxGR, poly (2,6-diphenylphenylene oxide), in order to provide a large surface area onto which the volatile chemicals can be trapped at ambient temperatures. When heating up the adsorbent with a flow of inert gas, the chemicals are released into the gas stream. The average concentration of chemicals in the mixture was 25 ng/pL.

Example 1 - Analysis of the complex mixture via the method of the invention using the apparatus of the invention

The apparatus of the invention is schematically presented in Figures 1 A and IB.

The test mixture (3 pL in a concentration of 25 ng/pL) was spiked onto adsorbent tube (1) loaded with TenaxGR; poly (2,6-diphenylphenylene oxide). The samples were run with three distinct chromatographic methods:

A) a traditional GC*GC technique;

B) one performing selective isolation (as per step d2’) the method of the invention), and

C) one performing subtractive isolation (as per step dl’) of the method of the invention). When using the method of the invention, performed with the apparatus of the invention

(cases A) and B)), the method parameters were identical except for the solenoid valve programming of the Deans switch (5). The Deans switch (5) was controlled by an electronic pressure controller (EPC) and a solenoid valve that was placed on the top of the GC oven (12), and the isolation line was passed through a flame ionization detector shaft (FID) with the jet removed. The temperature of the detector housing was kept at 300 °C to avoid the formation of cold spots. A Peltier cooler (11) comprising the adapter (12) as above described and represented in Figures 5A-C was attached to the FID port of the GC oven (13). The two different states of the Deans switch are presented in Figure 2.

A) First, a traditional GC*GC of the sample comprising: 2,6-dimethyl-4-heptanone (CAS: 108-83-8), 3-methyl cyclohexanone (CAS: 591-24-2), Methyleugenol (CAS: 93-15-2) and trans-P-Damascenone (CAS: 23726-93-4) 2-Pentanone and 1 -Butanol 2-butanol, 2- pentanone, 2-pentanol and methyl isobutyl ketone was introduced in the inlet (1) as thermal desorber (1). The primary (or tube) desorption was carried out using a flow rate of 35 mL min' ¹ at a temperature of 300 °C for 5 min. Meanwhile, the cold trap was kept at 5 °C. Upon completion, the trap was desorbed with a flow of 2 mL min'¹ at 300 °C for 7 min to ensure complete sample transmission into the GC*GC system. _The two columns (2 and 3) are placed inside the GC oven (13). The first column flow rate is 0.5 mL min-1, and the second column flow rate is 20 mL min-1 with a modulation period is 2 seconds. The GC oven (13) was set at an initial temperature of 40 °C and was kept there for 2 minutes. The temperature was ramped up to 260 °C with a rate of 3 °C min'¹ and kept there for 21 minutes. A first unprocessed two- dimensional total-ion chromatograms (TIC) of the original sample was obtained from the Agilent 7250 Q-TOF mass spectrometer and is shown in Figures 6. Figure 8A represents the total ion current (TIC) using the Q-TOF-MS detector (10) of an area of the full chromatographic spectrum of the standard mixture, wherein the selected standards, specifically 2,6-dimethyl-4- heptanone and 3-methyl cyclohexanone elute, and the zoomed area (Fig. 8A’) clearly indicates a constellation of closely eluting peaks, which cannot be divided nor even singularly analyzed, not even while using the very sensitive GC*GC setup. In fact, even in this two-dimensional chromatogram, the chemicals that appear at the same or overlapping “Retention time 7” values (peaks of 2,6-dimethyl-4-heptanone and 3-methyl cyclohexanone) are interfering with each other (co-eluting) (Figure 8 A and 8A’) and are only partially separated in the two-dimensional plane. _By changing the sequence of the ‘isolate’ and ‘divert’ states of the solenoid valve comprised in the Deans switch valve (5), two different types of isolation can be achieved according to the method of the invention.

Hence two further experiments were performed, where:

B) selective isolation, as per step d2’) of the method of the invention, was performed. Hence, the Deans switch (5) was programmed to follow the program as represented in Figure 3B. First, the Deans switch (5) and the solenoid valve therein comprised was set to convey the sample into the divert line d2) as represented in Figure 2B, thus applying to the pressure control valve (5’) of the Deans switch a pressure of 11.5 psi and a flow of the sample of 20 mL min'¹ is directed towards to the uncoated deactivated silica capillary (8) which is placed with an open end in the GC oven (13) and afterwards to the waste line (9), and thereby this segment of the chromatogram will not be visible by the Q-TOF -MS detector (10). Then, the Deans switch (5) is programmed to automatically switch to the isolation line dl) as represented in Figure 2A, and the flow is completely directed through the uncoated deactivated silica capillary (6) towards the passive splitter (7) which divides the flow towards: a transfer capillary (7a) towards a Q-TOF - MS detector (10) and an isolation capillary (7b) towards a recovery capillary (7c). The result of the TIC chromatogram in isolated mode is represented in Figure 6C. Figure 8C represents the total ion current (TIC) of an area of the full chromatographic spectrum of the standard mixture, wherein the selected standards, specifically 2-butanol, 2-pentanone, 2-pentanol and methyl isobutyl ketone elute, and the zoomed area (Fig. 8C’) clearly indicates that the peak overlapping, namely 2-Pentanone and 1 -Butanol, has been isolated and it is clearly visible, without any overlapping, previously present, as shown in Figures A- A’ .

C) subtractive isolation, as per step dl’) of the method of the invention, was performed. Hence, the Deans switch (5) was programmed to follow the program as represented Figure 3 A. First, the Deans switch (5) and the solenoid valve therein comprised was set to convey the sample into the isolation line dl) as represented in Figure 2A, and thus applying to the pressure control valve (5’) of the Deans switch a pressure of 11.5 psi_and a flow of the sample of 20 mL min'¹ is directed through the uncoated deactivated silica capillary (6) towards the passive splitter (7) which divides the flow towards: a transfer capillary (7a) towards a Q-TOF -MS detector (10) and an isolation capillary (7b) towards a recovery capillary (7c). Then, the Deans switch is programmed to automatically switch to the divert line d2) as represented in Figure 2B, and the flow is completely directed towards to the uncoated to the uncoated deactivated silica capillary (8) which is placed with an open end in the GC oven (13) and afterwards to the waste line (9), and thereby this segment of the chromatogram will not be visible by the Q-TOF -MS detector (10). In this case the Q-TOF-MS detector (10) will not detect the selected peak, while the rest of the sample is isolated, and therefore not visible on the two-dimensional chromatogram and left a “hole” in the chromatogram. The result of the TIC chromatogram in isolated mode is represented in Figure 6B. Figure 8B represents the total ion current (TIC) an area of the full chromatographic spectrum of the standard mixture, wherein the selected standards, specifically 2-butanol, 2-penanone, 2-pentanol and methyl isobutyl ketone elute, and the zoomed area (Fig. 8B’) clearly indicates that the 2-Pentanone and 1 -Butanol has been subtracted and left a hole without any overlapping, previously present, thus leaving the secondary peak visible. Figures 9A-B respectively represent the zoomed contour plots based on total ion current of an area of the full chromatographic spectrum of the standard mixture, wherein the selected standards, namely Methyleugenol and trans-P-Damascenone, elute (Fig.9A) where the peaks are clearly overlapping and merged (Methyl eugenol and trans-P- Damascenone), and the selectively isolated (Fig.9B) peak of Methyl eugenol. Moreover, Figures 7A and 7B show the 'residual'/subtracted (Fig. 6B) and 'isolated' (Fig. 6C) total ion chromatograms (TIC) (according to Example 2). In the zoomed image (Figure 7B) the time for the baseline stabilization is around 50 ms, indicating the fast stabilization of pressures due to the high flow supplied by the electronic pressure control module (EPC) that operates the Deans switch (5). This confirms that, because of this stabilization speed, it is possible to isolate peaks of a baseline width of 100 - 200 ms (such as the peak of 2-Pentanone and 1 -Butanol with respect to the peaks of Methyl eugenol and trans-P-Damascenone in Figures 9A-B.

Claims

1. A GCxGC apparatus for the selective isolation of a single or multiple compounds from complex mixtures comprising:

- a first column GC (2) and a second GC column (3) placed in a GC oven (13) with interposition of a flow modulator (4) in between;

- a first outlet (5a) connected through a capillary (6) to a passive splitter (7), apt to divide the flow of the vaporized liquid, gas or solid sample in a gas carrier into at least two lines, preferably two lines, a first transfer line comprising a transfer capillary (7a), directed to a Q-TOF- MS detector (10), and a second isolation line comprising an isolation capillary (7b), directed to a recovery capillary (7c) for the isolation of the sample;

2. The apparatus according to claim 1, wherein the sample injected via inlet (1) is a liquid sample comprising at least two co-eluting compounds, hence having at least partially overlapped GC peaks.

3. The apparatus according to anyone of claims 1 or 2, the first column (2) has a constant flow of the sample lower than the constant flow of the sample of the second column (3), preferably it is at least 10 times lower.

4. The apparatus according to anyone of claims 1-3, wherein, independently from one another:

- the first column (2) has a constant flow of the sample of 0.5 mL*min^-1 and

- the second column (3) has a constant flow of the sample of 20 mL*min^-1

5. The apparatus according to anyone of claims 1-4, wherein the valve (5) comprises pressure control point (5’) providing pressure of the carrier gas in the range from 10 to 20 psi.

6. The apparatus according to anyone of claims 1-5, wherein, independently from one another:

- the capillary (6) has dimensions of 0.1m * 0.25mm;

- the transfer capillary (7a) has dimensions of 3m * 0.18mm;

- the isolation capillary (7b) has dimensions of 0.8m * 0.25mm; and

- the capillary (8) has dimensions of 0.7m * 0.25mm.

7. The apparatus according to anyone of claims 1-6, wherein the cooling device (11) is a Peltier cooler device.

8. The apparatus according to anyone of claims 1-6, wherein the cooling device (11) is a Peltier cooler device comprising an adapter (12), which is preferably made of polycarbonate and comprising a heat insulating layer, preferably a heat insulating adhesive, more preferably having a thickness of 3mm and being made of condensation repelling polyvinyl chloride (PVC), a circular septum made of poly dimethyl siloxane having a diameter of 11 mm, and an O-ring having inner diameter of 5.92 mm and an outer diameter of 8 mm.

9. A method for identifying and/or separating analytes from a sample, comprising the steps of: a) providing a liquid, gas or solid sample to be analyzed; b) inserting the sample and a gas carrier into the heated inlet (1) of the apparatus according to anyone of claims 1-8, thus obtaining a vaporized sample; c) conveying the vaporized sample with a flow of carrier gas through a first column (2) and a second column (3), with interposition of a flow modulator (4) in between, such that the second flow rate for the second column (3) is higher than the first flow rate of the first column (2); d) conveying the sample from column (3) through a valve (5) into two alternative flow lines: an isolation line dl), wherein the sample flows from the valve (5) to the first outlet (5a), while the second outlet (5b) is closed, via a capillary (6), thus reaching, through a passive splitter (7), the Q-TOF-MS (10) through the transfer capillary (7a) and the recovery capillary (7c) through the isolation capillary (7b); or a divert line d2), wherein the sample flows from the valve (5) to the second outlet (5b), while the first outlet (5a) is closed, via a capillary (8), thus reaching the waste line (9); wherein the valve (5) is operated to switch the flow from isolation line (dl) to the divert line (d2); d) identifying and/or separating the analytes flowing from the isolation line dl).

10. The method of claim 9, wherein step d) comprises two alternatives:

11. The method according to anyone of claims 9-10, wherein the analytes comprise at least two co-eluting compounds.

12. The method according to anyone of claims 9-11, wherein the insertion of step b) is a liquid injection or a thermal desorption, more preferably a liquid injection, even more preferably it is selected from a hot split liquid injection and a solvent venting liquid injection.

13. The method according to anyone of claims 9-11, wherein the insertion of step b) is a thermal desorption, preferably it is a thermal desorption using as inlet (1) a thermal desorption device (1).

14. The method according to anyone of claims 10-13 for the isolation and separation from a complex mixture of single analytes, wherein the complex mixture is preferably a food or fragrances mixture.