NL2009010C2

NL2009010C2 - Process to separate compounds starting from a mixture of compounds.

Info

Publication number: NL2009010C2
Application number: NL2009010A
Authority: NL
Inventors: Jeroen Kool; Ferry Adam Henricus Heus
Original assignee: Stichting Vu Vumc
Priority date: 2012-06-15
Filing date: 2012-06-15
Publication date: 2013-12-17
Also published as: WO2013187761A1

Description

PROCESS TO SEPARATE COMPOUNDS STARTING FROM A MIXTURE OF

COMPOUNDS

The invention is directed to a process to separate compounds starting from a mixture 5 of two or more compounds, application of said process to perform gas chromatography fractionation and a system to perform the process.

Gas chromatography fractionation is described by F. Q. Yang, H. K. Wang, H. Chen, J. D. Chen, and Z. N. Xia, Journal of Automated Methods and Management in Chemistry, Vol. 2011(2011), Article ID 942467 (http://www.hindawi.com/journals/jamc/2011/942467/). 10 The article describes a process wherein volatile components are separated from the methanol extract of Curcuma rhizome. The compounds were separated on a stainless steel column packed with 10% OV-101 (3 m x 6 mm, i d ), and then, the effluent was split into two gas flows. One percent of the effluent passed to a flame ionization detector (FID) for detection and the remaining 99% were directed to the fraction collector. Five volatile compounds were 15 collected from the methanol extract of Curcuma rhizome (5g/mL) after 83 single re-injections (20pL) with the yield of 5.1-46.2 mg. The fraction collector directed the gaseous eluate from a gas chromatography column to 5 different sample collectors. Each supply to each sample collector was cooled with a dedicated cooling system and the sample collector comprised an organic solvent. In this manner the gaseous organic compounds as present in the gaseous 20 fractions as supplied to each sample collector will dissolve in the organic solvent. The structures of the isolated compounds were determined by their MS and NMR spectra.

A disadvantage of the above described gas chromatography fractionation is that only a limited number of 5 or 6 samples can be collected. A further disadvantage is that the fractionation process step is complex, requiring cold traps for every single fraction to be 25 collected.

The present process aims at providing a more simple and efficient process to separate compounds starting from a mixture of compounds. This is achieved by the following process.

Process to separate compounds from a mixture of two or more compounds by performing the following steps: 30 (a) passing a portion of a gaseous mixture of a carrier gas and the mixture of compounds through a column at an elevated temperature and wherein two or more compounds of the mixture are discharged at a different time from the column in a flow of discharged compounds and carrier gas, 2 (b) adding a fluid to the gaseous flow of carrier gas and discharged compounds as they are discharged from the column to obtain a flow comprising of the fluid, the carrier gas and the compounds, (c) cooling the flow comprising of the fluid, the carrier gas and the compounds to 5 obtain a flow comprising a gaseous phase comprising the carrier gas and a liquid phase comprising the fluid and the compounds, and (d) isolating one or more liquid fractions from the flow comprising the gaseous phase and the liquid phase, optionally after first separating the gaseous phase.

Applicant found that adding a fluid in step (b) and cooling in step (c) a liquid flow is 10 obtained which can be easily separated into fractions in a much simpler manner as compared to the prior art process. No separate cooling per fraction is required. This advantage makes it possible to apply isolation methods in step (d) which yield a high number of fractions. This is advantageous because a high degree of separation also referred to as a high resolution, can be achieved between the different compounds of the mixture of compounds. Further advantages 15 will be illustrated in the examples and described below when discussing the preferred conditions of the process according to the invention.

In step (a) a portion of a gaseous mixture of a carrier gas and the mixture of compounds is passed through a column at elevated temperatures. The column may be any column in which the different compounds of the mixture will have a different retention time 20 before they are discharged at the outlet of the column. The different retention times result from the fact that different compounds will interact differently with the inner surface of the column. By choosing a specific inner surface of the column better separation between the compounds may be achieved. In addition the length of the column, speed of the gaseous flow, and temperature will influence the degree of separation as is general knowledge to for 25 example the skilled person in the field of gas chromatography. The mixture of compounds may be injected onto a flow of carrier gas which flows through the column before the moment of injection. In step (a) two or more compounds of the mixture are discharged at a different time from the column in a gaseous flow of discharged compounds and carrier gas.

Suitably step (a) is performed by means of a gas chromatography separation. The 30 column is preferably a gas chromatography (GC) column. Such a column may be a glass, quartz glass or metal column provided with a suitable inner surface or film, referred to as stationary phase. Examples of suitable stationary phases are 5% phenyl, 95% dimethylpolysiloxane low bleed phase, high arylene modified phase and stabilized arylene-modified equivalent of a 35% phenylmethyl phase. Suitably polysiloxanes are used as a 3 stationary phase in the GC column. These polysiloxanes can have a range of functional groups to tune the polarity and thus the applicability of the compounds to be separated. For example, for very apolar compounds dimethyl syloxane may be applied. For more polar compounds, poly ethylene glycol is suitably used as the stationary phase. The internal 5 diameter of a capillary GC column is preferably between 0.1 mm and 0.32 mm. The length is suitably between 10 m and 100 m, with a stationary film thickness ranging suitably from 0.1 pm to 0.25 pm. If larger quantities of compounds are to be separated the column will suitably have a larger diameter, of for example 0.53 mm with a stationary phase film thickness of for example 5 pm and a length of for example 30 m. If very low boiling point compounds, such 10 as for example natural gas liquids, are to be separated it may be preferred to use packed columns, which are generally made of Pyrex glass or steel. Such a packed column may for example have a length of 1 to 5 meter and an internal diameter (i.d.) of up to 5 mm.

Preferably step (a) is performed at an elevated temperature. The temperature may be constant, a so-called iso-thermal separation, or more preferably start at a lower temperature 15 and rise according to a chosen gradient in temperature in time. The temperature in step (a) should be sufficient to evaporate the compounds to be separated in step (a). When applying a temperature gradient it may be that at the lower temperature range only the more volatile compounds of the mixture are in their gaseous phase and that at the higher temperature range also the less volatile compounds are in their gaseous phase. This may result in that the more 20 volatile compounds will be discharged earlier from the column than compared to the less volatile compounds. At the start of performing step (a) the temperature may be not sufficiently high to evaporate all or part of the fluid when the fluid is added in step (b). Suitably a fluid is used in step (b) which will either be supplied as a liquid or gas. The liquid fluid will evaporate at some stage at the higher temperature ranges of the temperature 25 gradient or at the isothermal temperature applied in step (a). The temperature in step (a) will in part be dependent on the compounds to be separated. Suitably a temperature or temperature gradient for step (a) is between 20 and 400 °C.

The elevated temperature in step (a) is suitably achieved by placing the column in a heated space, such as for example an oven. The heated space will transfer its heat to the 30 column to enhance the separation taking place in the column. The oven may suitably be a GC oven because these are readily available and because they are typically equipped with means to achieve and program a temperature gradient as described above. Any other heated space may also be used as long as the temperature in step (a) is achieved.

4

The pressure is suitably a pressure level suited for performing gas chromatography separation, suitably no special measures are taken to increase or reduce the pressure relative to the ambient pressure. The carrier gas may be any carrier gas known for use in gas chromatography, for example nitrogen, hydrogen or noble gasses such as helium.

5 Additionally, part of the flow of discharged compounds and carrier gas as obtained in step (a) can be analysed by an on-line gas chromatography detector by separating part of the flow before performing step (b) and analysing this separated part. In this manner a check is possible regarding the analysis of the different fractions as obtained in step (d). In step (b) a fluid is added to the gaseous flow of carrier gas and discharged compounds as they are 10 discharged from the column in step (a). The function of adding the fluid in step (b) is to introduce a condensable transport medium. The condensable transport medium will provide the liquid transport medium in which the separated compounds are dissolved in step (c). The temperature of the gaseous flow of carrier gas and discharged compounds as they are discharged from the column in step (a) and used in step (b) is preferably high enough to keep 15 the compounds in their gaseous phase. Because on average lower boiling compounds are discharged earlier than higher boiling compounds it may be that the temperature in step (b) can be lower at the start of the process and higher at the end of the process. Thus the preferred temperature of performing step (b) is between 20 and 400 °C, wherein the temperature may rise while performing step (b) along a temperature gradient or be constant, 20 isothermal. The temperature in step (b) may be about the same temperature as in step (a).

Step (b) may be performed in the same heated space as in which step (a) is performed. In that situation the temperature in step (b) will be about as in step (a). In a preferred embodiment step (b) is performed in the same oven and more preferably in the same GC oven as in which step (a) is performed. Optionally step (b) can be performed separately. Care should then be 25 taken that between performing step (a) and step (b) none of the compounds condense.

In step (b) a gaseous flow comprising of the fluid, the carrier gas and the compounds will be obtained, especially at the higher elevated temperatures of step (a). If a temperature gradient is applied it may happen that the fluid does not evaporate or fully evaporate at the lower temperatures of the applied temperature gradient as applied at the start of the process. 30 The fluid may be added in its gaseous phase or in its liquid phase. At the higher elevated temperatures in step (b) fluid added as a liquid may evaporate before, during or after contacting the fluid with the gaseous flow of carrier gas and discharged compounds.

The fluid added in step (b) can be any fluid which will result in a liquid flow under the conditions applied in step (c). Furthermore the compounds to be separated should 5 preferably dissolve in said liquid fluid. More preferably the fluid should have properties in that it can be simply removed from the one or more isolated liquid fractions as obtained in step (d). The fluid preferably is chemically inert with respect to the materials of the equipment used to perform the process according to the invention. Preferably the fluid 5 comprises an aprotic compound or mixture of aprotic compounds. Aprotic compounds are for example organic solvents which do not exchange protons with the compounds dissolved in it. Examples of suitable aprotic compounds are the below referred to paraffins and toluene. Preferably the fluid is a compound or mixture of compounds having a boiling point or T90vol% boiling range value in case of a mixture of below 200 °C. Examples of possible 10 fluids are water, acetonitrile, methanol, dimethyl ether, dimethyl sulfoxide (DMSO), and toluene and preferably paraffins, for example butane, iso-butane, iso-pentane, n-pentane, isohexane and/or n-hexane and mixtures thereof. Good results have been obtained using n-hexane, n-pentane and acetonitrile as the fluid.

Step (b) is suitably performed by supplying the fluid to the flow of discharged 15 compounds via a conduit which conduit is fluidly connected to an outflow conduit by means of a joint, for example a Y-joint or T-joint. Through this outflow conduit the flow of discharged compounds flows. The outflow conduit is fluidly connected to the discharge opening of the column used in step (a). Preferably the joint is located in the above described heated space. The joint may also be placed at the exterior of this heated space, provided that 20 the temperature of the gas as obtained in step (a) remains at the required elevated value to ensure that the compounds remain in their gaseous phase.

In step (c) the gaseous flow comprising of the fluid, the carrier gas and the compounds is cooled to obtain a flow comprising a gaseous phase comprising the carrier gas and a liquid phase comprising the fluid and the compounds. As explained above the 25 condensed fluid functions as a liquid transport medium in which the separated compounds are dissolved. The degree of cooling required in step (c) in order to condense the fluid will depend on the type of fluid. Thus preferably the gaseous flow comprising of the fluid, the carrier gas and the compounds are cooled to a temperature of between -80 °C and the boiling point of the fluid. For example, if butane is chosen as the fluid cooling to a lower temperature 30 range will be required as compared to when n-hexane is chosen as the fluid. In case n-hexane is used as the fluid it is found that cooling in step (c) to room temperature, wherein room temperature may be between 18 and 30 °C, or slightly above room temperature by means of indirect heat exchange is efficient. The gaseous flow is suitably cooled to a temperature of between -80 °C and room temperature. Cooling may be achieved by cooling the downstream 6 part of the transport conduit. Cooling of this downstream part is suitably performed by indirect heat exchange between the external surface of the conduit and a cooling medium.

The downstream part of the transport conduit is suitably the part of the transport conduit which is located externally of the above described heated space. Suitable cooling media are 5 dry ice, liquid nitrogen, chilled water, chilled air or ambient air or cooling bath compositions, such as for example slush bath compositions, or any peltier cooled metal. The cooling medium may be supplied along the exterior of the conduit in a current, counter-current or cocurrent manner, suitably the cooling medium is led through an annular space which runs for some distance along the exterior of the conduit and which space is provided with an inlet and 10 an outlet for cooling medium.

In step (a) a gaseous flow is obtained in which the compounds are separated because they are discharged by this flow at different times from the column. By performing steps (b) and (c) this separation of the compounds is maintained except in that the flow is liquid. The liquid fluid comprising the separated compounds can be considered to be an elongated 15 column of fluid or an elongated film of liquid along the interior of the column wherein along its length the separated compounds reside. By chopping this liquid column in parts one or more individual liquid fractions, enriched in one of the compounds relative to the composition of the starting mixture of compounds, are obtained. By enriched is here meant that the content of one compound has become higher relative to the content of one or more 20 other compounds in one isolated fraction obtained in step (d). These liquid fraction(s) can be easily stored for, for example, further analysis or purification. The liquid in the fractions can also be removed by evaporation to obtain the fractionated compounds in a more concentrated form. Such a process is much easier to perform with a liquid flow as compared to a gaseous flow. The present process thus combines the good separation performance often associated 25 with gas chromatography and the high product yield and the many possible collected fractions often associated with liquid chromatography.

In step (d) one or more liquid fractions enriched in one of the compounds is isolated from the flow comprising the gaseous phase and the liquid phase. The liquid fractions may comprise some gaseous phase. This gaseous phase, mainly comprising of the carrier gas, can 30 be easily separated from these fractions by a simple gas liquid separation. Suitably the gaseous phase is separated from the liquid phase before or during separating the liquid phase into its respective liquid fractions. For example, when the flow comprising the gaseous phase and the liquid phase are discharged into a collection space by means of pouring the gaseous phase may separate at the moment the flow leaves the discharge mouth. A simple and 7 preferred separation method is wherein the above described liquid column is cut or chopped in two or more liquid fractions. The number of different fractions will vary depending on the number of compounds to be separated. The number of different fractions may vary between 1 and 10000 and more preferably between 1 and 1000 and even more preferably between 20 5 and 400. The cutting or chopping can be performed by directing the liquid flow as it exists the transport conduit to separate sample collection spaces. By supplying the fluid to the different sample collection spaces one after the other each collection space will receive a different fraction of the above described elongated liquid column or film. Preferably the separate sample collection spaces are comprised in a so-called well plate. Well plates, also 10 known as Microtitre plate (spelled microtiter in the United States) or microplate or microwell plate, is a flat plate with multiple "wells" and are well known in the field of life sciences, screening laboratories, pharmaceutical laboratories, analytical research and clinical diagnostic testing laboratories. Examples of well plates suited for the present process have 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix. In this process the individual 15 wells are filled one after the other with the flow of the liquid phase, optionally in combination with the gaseous phase. Because separation may be very efficient in step (a) it could happen that part of the liquid column or film does not contain any compound. This will result in that some wells no compound is supplied to. In order to avoid that the compounds as separated into the different fractions evaporate it is preferred to cool the well plate for low boiling 20 compounds. For this, preferably the well plate is cooled or maintained at room temperature to between -80 °C and room temperature, which may be between 18 and 30 °C. The cooled well plate may be cooled elsewhere and removed from its cold storage and placed in the system according to the invention just before use. The well plate may also be continuously cooled during the process. The above process may advantageously be used to analyse a mixture of 25 compounds, suitably organic compounds, by first performing a process according to the invention and subsequently performing an analysis of the one or more fractions enriched in one of the compounds as obtained in step (d). Analysis of the fraction may be performed by well know analytical techniques like for example Mass Spectrometry (MS) or gas chromatography (GC) in combination with MS or with Nuclear Magnetic Resonance (NMR) 30 or with bio-assays.

A preferred analysis is a so-called bio-assay. In such a bio-assay the concentration, activity, affinity or effect of the separated compound or compounds can be determined by testing its effect on for example a living organism, on cells or on biochemical reagents or on proteins such as enzymes, receptors and antibodies or on DNA or on RNA. The invention is 8 therefore also directed to a process to perform a bio-assay on a mixture of compounds by performing the above process according to the invention, collecting the isolated fractions in separate wells of a well plate, evaporation of the fluid from the wells and performing a bioassay directly in the wells. Bio-assays can for example be performed on mixtures of mineral 5 oil compounds, mixtures of organic compounds comprising one or more fragrance compounds, mixtures of pharmaceutically active compounds, mixtures of food or food ingredients or mixtures of compounds derived from environmental samples polluted with unknown bioactives such as mixtures of polycyclic aromatic hydrocarbons (PAHs) and mixtures of pesticides.

10 Especially application in environmental chemistry is advantageous. The process according to the invention allows collection of a high number of fractions for further off-line bio-assay analysis by e.g. biological activity or affinity detection. This facilitates identification of dangerous biologically active pollutants in environmental samples. The process may be combined with already certified GC methodologies. This combination may 15 even itself become a new standard as many environmental pollutants are ideally separated by GC because of their physico-chemical properties. These compounds are often relatively low boiling and apolar compounds.

Other applications wherein the above bio-assay process may find a use are in the fragrances industry. Compounds separated by the process can be collected in a convenient 20 fashion for further off-line analysis techniques, such as bio-assays. The process may also find application in the food industry. Compounds separated by the process can be collected in a convenient fashion for further off-line bio-assay analysis techniques. For example, the process may be used for identifying which volatile compound or compounds act on e.g. nose receptors. This is relevant in the food industry as the smell of foods is a keystone to the food 25 industry. The process may also find application for analysing pheromones. Pheromones separated by the process according to the invention can be collected in a convenient fashion for further off-line bio-assay analysis techniques. This may be advantageous in the development of compounds, e.g. selective biological pesticides, that are able to lure insects, by means of insect traps and/or attract them, e.g. to pollinate selected plants in for example 30 greenhouses.

The invention is also directed to a system for separating a mixture of compounds. The system will be described with reference to Figure 1. The typical system comprises a column (3) having an inlet (2) for a compound mixture that can be injected via for example an auto injector (1) and a carrier gas and an outlet (8) fluidly connected to an outlet conduit (9), a 9 fluid supply conduit (7) fluidly connected to the outlet conduit (9) by means of a joint (4) and an indirect heat exchanger (ambient air for example) arranged to cool the outlet conduit (9), wherein the outlet conduit (9) is fluidly connected to a liquid separator (12). The preferred embodiment is seen in Figure 1 wherein column (3), outlet (8), part of outlet conduit (9) and 5 joint (4) are located in an oven (13).

Figure 1 also shows the preferred system wherein the liquid separator (12) comprises a fluid supply unit (6) and a well plate (11) comprising multiple wells and wherein the fluid supply unit (6) is operable to in time supply fluid to the multiple wells one after the other.

The process according to the invention is preferably performed in a system as 10 described above.

The invention shall be illustrated by means of the following non-limiting examples. Example 1 A mixture M containing twelve halogenated compounds in n-hexane was prepared.

15 The composition and concentration is described in Table 1. Lindane was used as standard (stock solution of 4,40* 10'2 M).

j Compound j ppmat~10'2M

20 :1) 2,2Dibromobiphenyl (2,2‘-DBB) [.................. 4,73 |

2) Fenchlorphos (FCP) j 4,87 J

3) Chlorpyrifos (CPP) j 5,31 J

4) Bromophos-methyl (B) j 5,54 j 5) cis-chlorfenvinphos (CFVP-a), and | 5,45 25 1 j 6) trans-chlorfenvinphos (CFVP-b) 7) 4,4'-Dibromobiphenyl (4,4-DBB) j 4,73 j 8) gamma-Cyhalothrin (CHT-a)and J 6,81 j 9) lambda-Cyhalothrin (CHT-b) j 10) cis-permethrin (PM-a) and 5,93 1 30 ! j 11) trans-permethrin (PM-b) | 12) Tralomethrin (TM) 10,1 j

............................................................................. .......................................J

Table 1 10

Step (a)

Mixture M was separated using a system as illustrated in Figure 1. The GC part of the system consisted of a Hewlett Packard (HP) 6890 series GC oven and a Hewlett Packard 5 6890 series auto injector all controlled by a PC containing HP GC ChemStation 7.0 software.

The injection port type was HP split/splitless and the port was equipped with all mm Agilent septum and a Restek Sky™ 4.0 mm ID Single Taper/Gooseneck Inlet Liner (4.0 x 6.5 x 78.5 mm). The column used was a FactorFour™ VF-5ms (5% phenyl / 95% dimethylpolysiloxane) 30 m x 0.37 mm x 250 pm I.D. capillary column. The column exit was 10 connected to a Restek Siltek® MXT® Y-Union Connector.

The injection volume of the above described mixture M was 1 pi and the GC injection port temperature was set to 300°C using splitless mode. The purging of the split vent was started 2.0 minutes after injection with 20 ml/min helium. The GC oven was programmed from 65°C (hold time 2.0 minutes) to 300°C (hold time 8.0 minutes) at 15°C/minute. Praxair 15 5.0 grade helium (Ylaardingen, The Netherlands) was used as ‘carrier gas’ and was kept at a constant column flow rate of 2.5 ml/min during the entire run, resulting in a headpressure of 170 kPa at 65°C oven temperature.

Step (b) 20 To the carrier gas (containing the gaseous analytes of mixture M) being discharged from the column, as described above, a continuous flow of n-hexane was provided, which served as fluid. The fluid was introduced to the Y-Union by a Harvard HA22I syringe pump equipped with a Hamilton GASTIGHT® 1005C-XP 5ml syringe, which was also fluidly connected to the Y-Union via a fluid transfer line (e.g. Restek Siltek® deactivated transfer 25 line (fused silica, I D. = 250 pm) The Y-Union was located in the oven as shown in Figure 1. During system optimization the flow rate of the fluid was varied at 84, 170, 340 and 700 pl/min. The carrier gas and the fluid mixed in the Y-Union. The resulting carrier fluid (containing the mixed-in carrier gas and possibly discharged compounds) was subsequently directed to the outlet conduit (Restek Siltek® deactivated transfer line (fused silica, I D. = 30 340 pm, length = 117 cm)). Via the outlet conduit (besides 340 pm I.D. also 250 pm I.D.

Restek Siltek® transfer line was tested), the carrier fluid was directed to the exterior of the oven.

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Step (c)

Once outside of the oven, the outlet conduit, and the herein flowing carrier gas and fluid, was cooled by indirect heat exchange between the ambient air (room temperature) and the exterior part of the outlet conduit (besides ambient air also solid carbon dioxide (CO2; -5 80°C) and solid COi/acetonitrile mixture (-40°C) was tested during system optimization).

The exit of the outlet conduit was connected to the XYZ arm of a fractionator. This fractionator was a modified Gilson 234 series auto injector, which was controlled by a PC and a Gilson 506C system interface. The temperature of the carrier fluid as discharged from the outlet capillary is about room temperature.

10

Step (d)

The mixed fluid, carrier gas and possibly discharged compounds were led via the outlet conduit to the fractionator (modified auto injector in the current setup) holding a polystyrene 96 micro titer well plate (Greiner bio-one CELLSTAR (Cat. No. 655180, Alphen 15 a/d Rijn, the Netherlands)), in an in-house built micro titer well plate holder. In the 96 well plate the fractions were collected with a pre-determined fractionation resolution of 30 seconds. The carrier fluid containing the separated analytes was deposited in the wells by a serpentine fractionation pattern. The fractionation resolution is determined by the time between that the exit of the outlet conduit is placed into a well and that the exit of the outlet 20 conduit is moved to the next well by the XYZ arm of the fractionator. The well plate was kept at room temperature.

In order to determine the recovery of the fractionated analytes of mixture M, the entire well plate was dried by a nitrogen flow. The fractions were subsequently re dissolved by 200 pi of solvent (n-hexane). This provides a nominal analyte concentration for all wells. The 25 dissolved fractions in solvent, containing the fractionated analytes, were transferred to GC vials. All fractions were subsequently analyzed by Gas Chromatography with an Electron Capture Detector (GC-ECD) for recovery. The peak areas of the analytes measured were corrected by using an internal standard (lindane) which was added directly after transferring the analytes from the well plate to the GC vials.

30 Figure 2 shows the recovery per compound of mixture M at varying fluid flow rates.

The fluid flow rate is defined as the amount of fluid exiting the outlet conduit during one minute (expressed in microliter/min) and is shown on the x-axis. The y-axis shows the relative recovery (%) per compound. Figure 2 lists the compounds in order of elution (the most upper compound being the first compound to elute from the column). The most upper 12 compound in the list corresponds to the most left placed compound in the graph per fluid flow rate and the remaining compounds are placed in the same order from left to right. The number codes used correspond to those of Table 1. The results show that a high recovery per compound is achieved in one analytical run for most compounds. From this experiment a 5 suitable fluid flow rate was determined to be 340 pl/min.

Example 2

Example 1 was repeated except that the carrier gas flow rate was varied while maintaining a fluid flow rate of 340 pl/min. The results are shown in Figure 3, presented in a 10 similar manner as in Figure 2. The carrier gas flow rate expressed in microliter/min (pl/min) is shown on the x-axis. The y-axis shows the relative recovery (%) per compound. From this experiment a suitable carrier gas flow rate was determined to be 2.5 ml/min.

Example 3 15 Example 1 was repeated with acetonitrile as the carrier fluid instead of n-hexane. The recovery found of the compounds was 90.4% ± 26.4%.

Example 4,

Example 1 was repeated with n-pentane as the carrier fluid instead of n-hexane and all carrier 20 fluid was collected during the GC fractionation run in one fraction. The recovery found of the compounds was 80.1% ± 27.3%.

Example 5

Example 1 was repeated with a different outlet conduit length and using a 25 fractionation resolution of 6.5 seconds. In this example the length of the outlet conduit was reduced to 54 cm instead of 117 cm as was used in Example 1. Similar results as to compound recovery were found as in Example 1.

Example 6 30 In this example a bio-assay analysis on fractionated benzo[a]pyrene (BaP) is conducted, using the process according to the invention, to evaluate the performance and applicability of the invention towards a possible application in the field of environmental chemistry. The bio-assay analysis was performed on polystyrene Greiner bio-one CELLSTAR (Cat. No. 655180, Alphen a/d Rijn, the Netherlands) well plates. The 96 well 13 plates were pretreated by applying DMSO onto the wells. This was performed by spotting 2.5 pi of 10% DMSO/water solution on the well plates by a Thermo Scientific Multidrop Combi nL. Hereafter the water of the spotted DMSO/water solution was allowed to evaporate under a nitrogen flow. After this pretreatment, solutions containing BaP (1000 ppm, 200 ppm, 40 5 ppm and 8 ppm) as obtained from Acros Organics (Geel, Belgium) were fractionated in a similar manner as steps (a)-(d) illustrated in Example 1 using n-hexane as carrier fluid. The fraction resolution in step (d) was 6.0 seconds/well. Afterwards, internal calibration standards were added as 1, 10, 100 and 1000 ppm BaP in hexane to known wells on the plates. The whole plates were then dried under nitrogen and stored at -20°C until the bio-assay was 10 performed.

Dioxin like activity was measured using stably transfected H4IIE cells according to M. Machala, J. Vondracek, L. Blaha, M. Ciganek and J. Neca “Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using an in vitro reporter gene assay.” Mutation research, vol. 497, nr. 1-2, pp. 48-62, 2001, with 15 modifications. For this cells were cultured in aMEM (Gibco, The Netherlands) supplemented with 10% FBS (Sigma, Germany) and Penicilline/Streptomycine (Gibco, The Netherlands). Prior to exposure, cells were suspended in medium for transfer to the well plates.

The well plates as obtained above were directly filled with the thus obtained cell suspension (100 pi per well). In this way, cells are directly exposed to the fractions. 20 Normally, fractions dissolved in for example DMSO are added to cells for bio-assays. In the current setting, cells are added directly to the fractions and allowed to attach to the well plates while being exposed simultaneously. This circumvents additional transfer steps and prevents dilution of the collected fractions allowing simpler, faster and more sensitive analysis. 48 hours after exposure, cells were visually checked for cytotoxic effects. After that, cells were 25 lysed and measured for luciferase activity according to the above referred method according to Machala et al. The activities found were plotted against retention time per fraction collected to obtain bioactivity chromatogram data. The results are shown in Figure 4.

This example illustrates that a bio-assay can be performed in a very simple and straightforward manner after GC fractionation in the wells of a well plate. BaP shows tailing 30 in the results. This always occurs for BaP separated under the conditions used. This is also observed when performing standard GC-Flame Ionization Detection under similar chromatographic conditions.

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Example 7

Example 1 was repeated with a fractionation resolution of 6.5 seconds. Now for demonstration of fractionation resolution only the following fractionated compounds, in the respective fractions were they were collected, are shown in Figure 5a when using 6.5 sec 5 fractions: 2,2'-DBB, FCP and CPP. The recovery found of all compounds averaged was 71.9% ±23%.

Now for demonstration of fractionation resolution, only the following compounds were collected with 3.0 sec fractions: 2,2'-DBB, FCP and CPP. The recovery found of the compounds was 58.7% ± 3.6%. The results are shown in Figure 5b.

10 Compounds were collected in one or two fractions demonstrating the maintenance of resolution after GC separation.

Claims

Method for separating substances, starting from a mixture of two or more substances, by performing the following steps: a) passing a part of a gaseous mixture of a carrier gas and a mixture of substances through a column at an elevated temperature, and wherein two or more substances of the mixture are discharged from the column at a different time in a flow of discharged substances and carrier gas, b) adding a fluid to the gaseous flow of carrier gas and discharged substances, when they are discharged from the column, in order to obtain a flow comprising the fluid, the carrier gas, and the substances, c) cooling the flow comprising the fluid, the carrier gas, and the substances, in order to obtain a flow which comprises a gaseous phase comprising the carrier gas, and a liquid phase comprising the fluid and the substances, and d) isolating one or more liquid fractions from the flow comprising the gaseous phase and the liquid phase, optionally after having first separated the gaseous phase.

The method of claim 1, wherein step a) is performed in a gas chromatography column disposed in a heated space.

3. Method as claimed in claim 1, wherein step b) is carried out by adding the fluid to the flow of discharged substances via a conduit, said conduit being in fluid communication via a connection with a discharge conduit via which the flow of the discharged substances flows.

4. Method as claimed in any of the claims 1-3, wherein the fluid that is added in step b) is a liquid or gas-shaped fluid, and wherein, in the case that a palpable fluid is added, the fluid fluid in step b) evaporates before, during, or after coming into contact with the fluid with the gaseous flow of carrier gas and discharges.

5. A method according to any one of claims 1-4, wherein the temperature in step a) is between 20 and 400 ° C.

The method of any one of claims 1-5, wherein the temperature in step b) is between 20 and 400 ° C.

Method according to any of claims 1-6, wherein the mixture of substances is a mixture of mineral oil components, fragrances, aromas, components of perfumes, pheromones, biologically active substances, food odors and / or environmental samples such as contaminated environmental samples containing PAHs and / or pesticides.

A method according to any one of claims 1-7, wherein the fluid added in step b) is a substance or a mixture of substances with a boiling point or with a T90vol% boiling range in the case of a mixture of substances that is lower than 200 ° C.

9. A method according to claim 8, wherein the fluid comprises an aprotic compound or a mixture of aprotic compounds.

The method of any one of claims 1-9, wherein the gaseous flow comprising the fluid, the carrier gas, and the substances is cooled in step c) to a temperature at which the fluid will condense. 25

The method of claim 10, wherein the gaseous flow comprising the fluid, the carrier gas, and the substances is cooled to a temperature comprised between -80 ° C and the boiling point of the fluid.

A method according to any one of claims 1 to 11, wherein step d) is carried out by applying the one or more liquid fractions enriched in one of the substances in the course of time to one or more separate sample collection spaces.

The method of claim 12, wherein different isolated fractions are provided in between 10 and 10,000 different collection spaces.

14. Method as claimed in any of the claims 12-13, wherein the different sample collection spaces are provided in containers, such as a titer plate, and wherein the individual containers are filled one after the other with the flow comprising the gaseous phase and the liquid phase, optionally with substances and possibly after first separating the gaseous phase.

The method of claim 14, wherein the containers are cooled to a temperature comprised between -80 ° C and room temperature, wherein room temperature is a temperature comprised between 18 and 30 ° C.

16. A method according to any one of claims 1-15, wherein after step d) the fluid is removed from the one or more liquid fractions enriched in one of the substances, and this by evaporating the fluid and by separating the evaporated fluid.

17. Method for analyzing a mixture of substances, first by carrying out a method according to any of claims 1-16, and then by performing an analysis of the one or more fractions enriched in one of the substances, as obtained in step d).

The method of claim 17, wherein the analysis of the one or more fractions enriched in one of the substances as obtained in step d) is a bioassay. 25

A method of performing a bioassay on a mixture of substances, by performing the method of claim 18, wherein the isolated fractions in step d) are collected in separate wells of a titer plate, removing the fluid from the wells evaporation, and performing a bio-assay directly in the wells. 30

A method according to claim 19, wherein the mixture of substances is a mixture of polyaromatic hydrocarbons (PAHs), mixtures of pesticides, mixtures of mineral oil components, mixtures of organic substances comprising one or more fragrance substances, mixtures of pharmaceutically active substances , mixtures of food ingredients, or mixtures of substances derived from environmental samples contaminated with bioactive compounds. 5

A system for separating a mixture of substances, comprising a column provided with a feed for a mixture and a carrier gas, and with a drain that is in fluid communication with a drain line, wherein a fluid feed line is in fluid communication with the drain line through by means of a connection, as well as an indirect heat exchanger arranged to cool the discharge line, the discharge line being in fluid communication with a fractionating unit.

The system of claim 21, wherein the column, the drain, a portion of the drain line, and the connection are arranged in a heated space. 15

A system according to any one of claims 21-22, wherein the fractionating unit comprises a titer plate comprising a plurality of wells, and wherein the system is also provided with a fluid supply unit which is operative to successively add to the plurality of wells fluid over time. to add. 20

A method according to any one of claims 1-20, as implemented in a system according to any one of claims 21-23.