DE102007056670A1 - Apparatus for separation/analysis of isomer compounds has a structured connection between the gas chromatograph and the nuclear magnetic resonance measurement cell - Google Patents

Abstract

The assembly to separate and analyze the constituents of complex chemical samples, especially entantiomers of stereo isomers, has a gas chromatograph (4), a nuclear magnetic resonance spectroscope (7) and a connection (6) between them. The ratio between the inner diameter of the gas chromatograph outlet and the inner diameter of the entry into the connection and/or the ratio between the inner diameter of the connection outflow and the inner diameter of the entry into the nuclear resonance measurement cell is a maximum of 4.

Description

The The invention relates to a method and a device for detecting complex chemical mixtures.

According to the state of the art, mixtures of substances have long been routinely investigated by combining gas chromatography devices with mass spectrometry devices. Accordingly, there are a large number of reviews ( Steiner, S .; Huber, C. News from Chemistry 51, 2003, 387-389 ; Tomer, KB; Moseley, MA; Deterding, LJ; Parker, CE Mass Spectrometry Reviews, 1994, 13, 431-457 ; Lee, MS; Kerns, EH Mass Spectrometry Reviews, 1999, 18, 187-279 ). Although this methodology provides information on the number of individual components and their assignable molecular weights, to date no continuous method is known which at the same time provides concrete information about the chemical structure of these individual components.

Generally However, substances of the same molecular formula become more different Structure referred to as constitutional or structural isomers. If Atoms of a substance are similar, but different possess spatial orientation, one speaks of stereoisomers. To the stereoisomers include the configuration isomers and Conformational isomers. The configuration isomers are I am geometric isomers such. As the cis / trans or E / Z isomers. in principle is a conversion to another isomer for configuration isomers only by loosening and reconnecting bonds possible. Conformational isomers, however, by Turn rotation around single bonds without binding solution into each other.

A particularly important group of stereoisomers are the enantiomers (optical isomers). They behave like incongruent (non-coincidental) Image and reflection and one speaks of chiral molecules. Both enantiomers together are referred to as racemate mixture.

A Another important type of stereoisomers are the diastereomers. Diastereomers are stereoisomers that are not enantiomers and thus do not behave like an incongruent image and mirror image. Diastereomers can be chiral or achiral.

Generally can be found in the literature even more names, the intramolecular spatial arrangement of atoms describe. So are the names erythro and threo (or like and unlike) common for molecules that identical or different substituents on two stereogenic centers wear. The two are identical or similar substituents at the Fischer projection on the same page, so will this Configuration isomer as meso (same substituents) or erythro form (unterschieldliche Substituents). Are the substituents in the Fischer projection on different sides, this forms configuration isomer the threo form. These examples are illustrated by the tartaric acids (meso form, racem form) and at the sugar molecules D (-) - erythrose and D (+) - threose.

Also a common description for a substituent position for stereoisomers, the endo and exo position of substituents such as For example, bicyclo [2.2.1] heptane. At this connection will thus distinguish whether there is a substituent on the side of the methylene bridge (exo) or on the side of the ethylene bridge (endo). Should this compound be unsaturated on an ethylene bridge Thus, the two substitutable positions of the methylene bridge must be be distinguished as well. This is called the position which points to the side of the double bond, as syn position. The Position pointing to the opposite side becomes anti-position called.

It should be noted that stereoisomerism, with its various special cases, has acquired particular importance in research, since physiological effects often depend on the immediate structure rather than on the composition of an active substance ( Fränkel, S. Monatsschrift paediatrics 1904, Volume 3, Number 1, 290-308 ).

For this reason, as early as 1981, first experiments by Buddrus and Herzog were published, which deal with the coupling of gas chromatography (abbreviated: GC) with nuclear magnetic resonance spectroscopy (abbreviated: NMR) ( Organic Magnetic Resonance, 15, no. 2, 1981, 211-213 ). Relatively large-volume, mutually uncoordinated and very unspecific arrangements were used, which allowed only the investigation of structurally very different compounds, such as mixtures of diethyl ether, 2,2-dimethylbutane and cyclopentane. Another disadvantage is the high time required per detected substance, which results from the long duration of the individual measurements and their accumulation.

Later, further attempts were made to circumvent these disadvantages ( MD Grynbaum et al., Analytical Chemistry, 79, No, 7, 2007, 2708-2713 ). Compared to Buddrus and Herzog continuous investigations were carried out here, the device arrangement used could ever but also only structurally very different compounds such. B. separate a mixture of diethyl ether, tetrahydrofuran and dichloromethane and detect simultaneously.

The Object of the present invention is therefore to overcome the disadvantages of known methods and devices to circumvent and a method and to provide a device with which at a low Time requirement continuously isomeric compounds are detected can.

These Task is achieved by a device according to claim 1 solved. Preferred embodiments and developments this device and the associated method and Use are described in claims 2 to 20.

The device according to the invention consists of three essential components: a gas chromatography device, a nuclear magnetic resonance spectroscopy device and a connector which connects the two devices ( 1 ). It has surprisingly been found that for the solution of the above object according to the invention is of crucial importance, how large the ratio between the inner diameter of the outlet of the gas chromatography and the inner diameter of the inlet of the connector, and the ratio between the inner diameter of the connector and the inner diameter of the Inlet in the nuclear magnetic resonance cell is. It could be shown that these differences in the inner diameters at these two points of connection should be as small as possible. According to the invention, the ratio of the inner diameter of the outlet of the connector to the inner diameter of the inlet of the nuclear magnetic resonance cell and the ratio of the inner diameter of the outlet of the gas chromatographic device to the inner diameter of the connector is at most 4, preferably smaller 2. In particular, this difference is 10%. In a particularly preferred embodiment, the inside diameters of the outlet of the gas chromatography apparatus and the inlet of the connector and the inside diameters of the outlet of the connector and the inlet of the nuclear magnetic resonance cell are the same within the measurement error.

In An embodiment of the invention is the connector formed in the form of a transfer capillary. This transfer capillary is attached to the nuclear magnetic resonance cell such that the nuclear magnetic resonance cell facing opening of the transfer capillary and the opening the nuclear magnetic resonance cell as large or allow undisturbed sample flow.

In An alternative embodiment of the invention becomes the function of the connector from the capillary separation column taken over by the gas chromatograph. In this case will without the use of an additional transfer capillary the Capillary of the gas chromatograph directly to the supply line to the nuclear magnetic resonance spectroscopy device or connected to the input capillary of the nuclear magnetic resonance cell. This design can, for. B. be advantageous if the stray field of the magnet of the nuclear magnetic resonance spectroscopy device no interference caused.

The used in the device according to the invention Nuclear resonance spectroscopy device preferably has the measurement frequency of more than 60 megahertz, preferably more than 100 megahertz, especially more than 300 megahertz. Preference is given to devices used with non-rotating probe head.

at Although the gas chromatography apparatus according to the invention can be relatively large-volume such. B. preparative gas chromatography columns This would, however, at the output of the column a Require transfer capillary. Preference is however semi-preparative or packed gas chromatography columns, wherein also here at the outlet of the column a transfer capillary required is. In the choice of transfer capillary should in particular to the o. g. Tolerance width with respect to the inner diameter of the rest used capillaries or the supply line in the NMR measuring cell are respected.

at small amounts of sample come commercially available or self-filled gas chromatography capillaries are used. The on the gas chromatography capillaries amount of sample to be charged in this case, a maximum of 5 mg, preferably a maximum of 1 mg, in particular maximum 100 μg.

Furthermore, the invention includes the flow-through sample head required for the device according to the invention. The basic components of the probe used ( 2 ) are a U-tube, which is preferably made of glass, which is connected at one or both ends, each with a capillary connector, and a measuring coil. The capillary connector ensures the transfer of the gas from the transfer capillary or the outlet of the GC capillary separation column into the actual measuring cell of the nuclear magnetic resonance spectroscopy apparatus.

In the known from the prior art capillary (capillary), it is not provided according to the previously valid problem that the capillary to be connected can be replaced, so the attachment of the capillary by means of simple gluing takes place. For the ge in the present invention The usual capillary connectors are less suitable because the capillaries can not be exchanged there. Therefore, a special capillary connector has been developed for the device according to the invention, in which an exchange of capillaries is possible. For this purpose, the capillary connector is provided with recesses at both ends into which the capillary guides can be inserted or screwed. The transfer capillary suitable for the particular problem can now be fixed in the capillary guide. By replacing the transfer capillary, the inner diameter of the transfer line can be changed without much effort.

Of the Flow of the used for the chromatographic separation process mobile phase is to be chosen so that the length of stay of a molecule in the detection cell the period of time for a single nuclear magnetic resonance spectroscopic measurement is needed, not below. When falling short the duration of a single nuclear magnetic resonance spectroscopic measurement the signal intensity of the spectrum goes to zero and a detection is therefore no longer useful. For example, would be for a sample head with an active detection volume of 1.5 μl and a measurement period of a single nuclear magnetic resonance spectroscopic Measurement of 0.2 s the flow rate 0.2 ml / min makes the most sense.

The Flow rate moves according to the invention between 0.01 ml / min and 50 ml / min, preferably between 0.1 ml / min and 5 ml / min, more preferably between 0.1 ml and 1 ml.

In a preferred embodiment, the inventive Device heating means on which the sample-carrying components Heat (gas chromatography capillaries, transfer capillary, detection cell) can. It is preferred that the temperature of the sample-carrying Components between 0 ° C and 300 ° C, preferably between 10 ° C and 150 ° C, in particular between 20 ° C and 50 ° C is.

The Use of heatable sample components such as the column oven as well as the transfer capillary and the probe head has the advantage that even less volatile substances such as B. monoterpene, sesquiterpenes and diterpenes in the gas phase be enriched. An investigation of a complex mixture of substances, from Z. As terpenes, by means of the device arrangement described is possible only with the help of a heating system, as the vapor pressure, and thus the concentration in the gas phase, is too low at Raumtemperautur.

One great advantage of the device according to the invention and the method is that in addition to structural isomers Also stereoisomers can be separated and clearly identified. The The device and the method even allow the separation a racemate mixture in the individual enantiomers and their detection. In addition, by an extremely short admission of the measuring signal combined with a high pulse repetition rate a fast accumulation the spectra is enabled and thus a sensitivity gain results in a spectral uptake of smaller amounts of sample allows. This not only reduces the detection limit but are studies of the smallest sample mixtures of well below 5 .mu.mol per component possible because also the chromatographic resolution of the signals significantly increases. For a molecule like diethyl ether The specification of 5 μmol, for example, corresponds to an analysis quantity of 0.1 μl.

Further Advantages, features and applications of the invention will be described below with reference to the embodiments described below described with reference to the drawings. In the drawings show:

1 : Schematic representation of the device according to the invention: 1 Gas bottle with carrier gas (eg nitrogen); 2 Flow controllers; 3 Injection valve; 4 gas chromatograph; 5 Column oven; 6 Connector (in this example, a transfer capillary); 7 NMR spectrometer;

2 : Schematic representation (not to scale) of a solenoid capillary flow-through sample head according to the invention: 1 Universal capillary connector (capillary holder); 2 Glass U-tube (about 0.8 mm inside diameter); 3 Measuring coil; 4 Transfer capillary (outer diameter 0.36 mm, inner diameter variable); 5 Capillary guide for the transfer capillary (eg PEEK screw with hole); 6 Capillary guide (eg PEEK-Schrabube with hole);

3 : 2 D plot of the GC-NMR experiment of 0.1 μl diethyl ether, 0.2 μl 1,2-cis-dimethylcyclohexane and 2 μl 1,2-trans-dimethylcyclohexane; the box marked with a square indicates the area in which a measurement signal for the substances contained in the sample would be expected; in this measurement, however, the signal remains off;

4 : 2 D plot of the GC-NMR experiment of 0.8 μl diethyl ether, 0.4 μl 1,2-cis-dimethylcyclohexane and 0.4 μl 1,2-trans-dimethylcyclohexane;

5 : 2 D plot of the GC-NMR experiment of 0.1 .mu.l of diethyl ether, 0.2 .mu.l of 1,2-cis-dimethylcyclohexane and 0.2 .mu.l of 1,2-trans-dimethylcyclohexane.

embodiments

For the first component of the device according to the invention can be commercially available nuclear magnetic resonance spectroscopy equipment For example, the manufacturer Varian (Varian Germany GmbH, Darmstadt, Germany) or Bruker BioSpin GmbH (Rheinstetten, Germany) be used. Thus, in the present invention the Bruker UltraShield 400 MHz and Bruker devices UltraShield Plus 700 MHz successfully used.

The nuclear magnetic resonance imaging apparatus can be equipped with a specially prepared solenoid probe head ( 1 ). Such a solenoid capillary flow-through sample head is characterized in that the measuring coil is wound directly around the measuring cell and the detection capillary itself lies at right angles to the basic magnetic field B _{0 of} the cryomagnet.

The Detection volume of the measuring cells is usually below of 50 μl. However, it is preferable to use detection volumes less than 5 μl, in particular less than 1.5 μl.

The construction arrangement with respect to the measuring coil on which the probe head is based is described in the article ( TL Peck, RC Magin, PC Lauterbur, J. Magn. Resn, Series B 108, 1995, 114-124 ).

Exactly but also commercially available sample heads of the type Proton MicroFlow NMR Probe (Protasis, Marlboro, USA) become.

For the second component of the device according to the invention can be commercially available gas chromatographs z. B. the companies Agilent Technologies (Agilent Technologies Germany GmbH, Böblingen, Germany) or Varian / Chrompack (Varian Germany GmbH, Darmstadt, Germany).

Columns used for this purpose can themselves be occupied or can be obtained commercially (eg from the companies Ziemer GmbH (Langerwehe, Germany) or Restek (Bad Homburg, Germany)). Commercially available types of columns are for example: Rx ^TM -1 ms Columns (fused silica, cross Bond ^® 100% dimethyl polysiloxane), Rx ^TM 5ms Columns (fused silica, cross Bond ^® dipheny 5% / polysiloxane 95% dimethyl, Rtx ^® -1 Columns (fused silica, cross Bond ^® 100% dimethyl polysiloxane), FS-Cyclodex beta-I / P (Permethyl-β-cyclodextrin), SC -101-NB (Methylsilicon, fluid) All of these columns are available with a capillary inside diameter of 250 microns and 320 microns and a length of 15 m-60 m at the above-mentioned chromatographic specialist dealers In addition to these indicated capillary inside diameters and Column lengths are also other dimensions commercially available.

A suitable column combination is for example a Chirasil beta Dex Column for the gas chromatograph (25 m, 250 μm i.d., 0.25 μm film thickness) combined with a fused silica Transfer capillary (2 m, 250 μm i.d.) and one into the flowhead Glued fused silica capillary (0.5 m, 250 μm i.d.). The above-mentioned tolerance factor is within the measurement accuracy according to the manufacturer's instructions in this device arrangement the same 0th

Around the advantages of the device according to the invention Compared to the prior art, the Measurements in the first two examples after the from the state of Technique known method, and in the third example according to the invention Procedure performed. Except the different one Ratio of the inner diameter of the gas chromatography column and the transfer capillary, the other parameters were identical.

example 1

In this example, a device arrangement according to the prior art was used ( according to MD Grynbaum et al., Analytical Chemistry, 79, No, 7, 2007, 2708-2713 ).

Equipment:

• NMR spectrometer: Bruker ARX 400 (Bruker BioSpin GmbH, Rheinstetten, Germany)
• Gas Chromatograph: Fractovap Series 2350 (Carlo Erba Strumentazione, Milan, Italy)

Gas chromatography column:

• Separation Phase Chirasil-beta-Dex
• Length: 25 m
• Inner diameter: 250 μm
• Film thickness: 0.25 μm

transfer capillary:

• Length: 2 m
• Inner diameter: 50 μm

Sample volume:

• 0.2 μl of 1,2-trans-dimethylcyclohexane, 0.2 μl of 1,2-cis-dimethylcyclohexane, 0.1 μl of diethyl ether

Measurement parameters:

• Column temperature: 60 ° C
• Number of measurement series: 128 rows
• Number of transients per row: 64
• Total measuring time: 30 min
• Transient duration: 0.22 s

3 clearly shows that the device arrangement is not suitable for separating and detecting cis and trans isomers. The concentration of the analytes in the measuring cell is too low with this device arrangement, so that the detection of the individual components is not possible.

Example 2

In this example, a device arrangement according to the prior art is used ( by MD Grynbaum et al., Analytical Chemistry, 79, Nr, 7, 2007, 2708-2713 ), analogous to the device arrangement of Example 1, but the amount of injected substance was increased.

Equipment:

• NMR spectrometer: Bruker ARX 400 (Bruker BioSpin GmbH, Rheinstetten, Germany)
• Gas Chromatograph: Fractovap Series 2350 (Carlo Erba Strumentazione, Milan, Italy)

Gas chromatography column:

• Separation Phase Chirasil-beta-Dex
• Length: 25 m
• Inner diameter: 250 μm
• Film thickness: 0.25 μm

transfer capillary:

• Length: 2 m
• Inner diameter: 50 μm

Sample volume:

• 0.4 μl of 1,2-trans-dimethylcyclohexane, 0.4 μl of 1,2-cis-dimethylcyclohexane, 0.8 μl of diethyl ether

Measurement parameters:

• Column temperature: 60 ° C
• Number of measurement series: 128 rows
• Number of transients per row: 64
• Total measuring time: 30 min
• Transient duration: 0.22 s

4 shows that the increase in the amount of substance injected allows the detection of the more volatile component (diethyl ether). However, it can be observed that the elution time of the peak increases to more than 10 minutes and thus a separation of two isomers would not be possible. The detection of the less volatile 1,2-cis / trans dimethylcyclohexanes is not possible despite the increased injection quantity.

Example 3

This Example shows a device arrangement according to the invention, at the inner diameter of the gas chromatography column and the transfer capillary are the same.

Equipment:

• NMR spectrometer: Bruker ARX 400 (Bruker BioSpin GmbH, Rheinsteffen, Germany)
• Gas Chromatograph: Fractovap Series 2350 (Carlo Erba Strumentazione, Milan, Italy)

Gas chromatography column:

• Separation Phase Chirasil-beta-Dex
• Length: 25 m
• Inner diameter: 250 μm
• Film thickness: 0.25 μm

transfer capillary:

• Length: 2 m
• Inner diameter: 250 μm

The Transfer capillary was placed in the capillary connector using a PEEK fittings and a tubing sleeve bolted.

Sample volume:

0.2 μl 1,2-trans-dimethylcyclohexane, 0.2 μl of 1,2-cis-dimethylcyclohexane, 0.1 μl of diethyl ether

Measurement parameters:

• Column temperature: 60 ° C
• Number of measurement series: 128 rows
• Number of transients per row: 64
• Total measuring time: 30 min (imaged measuring time: 13 min)
• Transient duration: 0.22 s

As the 5 shows, with the inventive device consisting of the above-mentioned gas chromatograph with built-GC column, a transfer capillary and a nuclear magnetic resonance spectroscopy device, cis-trans isomers can be identified with a short analysis time.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• Steiner, S .; Huber, C. News from Chemistry 51, 2003, 387-389 [0002]
• - Tomer, KB; Moseley, MA; Deterding, LJ; Parker, CE Mass Spectrometry Reviews, 1994, 13, 431-457 [0002]
• - Lee, MS; Kerns, EH Mass Spectrometry Reviews, 1999, 18, 187-279 [0002]
• - Fränkel, S. Monatsschrift paediatrics 1904, Volume 3, Number 1, 290-308 [0008]
• - Organic Magnetic Resonance, 15, no. 2, 1981, 211-213 [0009]
• MD Grynbaum et al., Analytical Chemistry, 79, No, 7, 2007, 2708-2713 [0010]
• TL Peck, RC Magin, PC Lauterbur, J. Magn. Resn, Series B 108, 1995, 114-124 [0035]
• according to MD Grynbaum et al., Analytical Chemistry, 79, No, 7, 2007, 2708-2713 [0041]
• according to MD Grynbaum et al., Analytical Chemistry, 79, No, 7, 2007, 2708-2713 [0043]

Claims (20)

Device for separation and analysis of complex chemical samples consisting of a gas chromatography device, a nuclear magnetic resonance imaging apparatus and a connector which the gas chromatography apparatus and the nuclear magnetic resonance spectroscopy apparatus connects the relationship between the Inner diameter of the outlet of the gas chromatography device and the inner diameter of the inlet of the connector and / or the ratio between the inner diameter of the Outlet of the connector and the inner diameter of the inlet of the nuclear magnetic resonance cell is 4 at maximum. Apparatus according to claim 1, wherein the ratio between the inside diameter of the outlet of the gas chromatography apparatus and the inner diameter of the inlet of the connector and / or the ratio between the inner diameter of the Outlet of the connector and the inner diameter of the inlet of the nuclear magnetic resonance cell is 2 at maximum. Device according to one of the preceding claims, the ratio between the inner diameter of the Outlet of the gas chromatography device and the inner diameter the inlet of the connector and / or the ratio between the inner diameter of the outlet of the connector and the inner diameter of the inlet of the nuclear magnetic resonance cell maximum 10%. Device according to one of the preceding claims, the ratio between the inner diameter of the Outlet of the gas chromatography device and the inner diameter the inlet of the connector and / or the ratio between the inner diameter of the outlet of the connector and the inner diameter of the inlet of the nuclear magnetic resonance cell within the measuring error is maximum 1. Device according to one of the preceding claims, wherein the connector is formed as a transfer capillary is. Device according to one of the preceding claims, wherein the connector on the nuclear magnetic resonance cell is such is fixed, that its the nuclear magnetic cell facing opening and the opening of the nuclear magnetic resonance cell as possible allow undisturbed sample flow. Device according to one of the preceding claims, wherein the connector is the capillary separation column of the gas chromatography apparatus. Device according to one of the preceding claims, furthermore with heating means for heating the gas chromatography capillaries, the connector and / or the detection cell. Device according to one of the preceding claims, wherein the nuclear magnetic resonance spectroscopy apparatus is the measurement frequency of more than 60 megahertz, preferably more than 100 megahertz, especially more than 300 megahertz. Device according to one of the preceding claims, wherein the nuclear magnetic resonance spectroscopy apparatus is a non-rotating Sampling head has. Flow-through sample head for use in a device according to one of the preceding claims, consisting of a U-tube, which at one or both ends with each a capillary connector, and a measuring coil, wherein the capillary connector for exchanging capillary wells having plug or screw connections at both ends. Capillary connector for use in a device according to one of the preceding claims, with recesses with plug or screw connections at both ends. Process for the separation and analysis of complex chemical Samples by means of a device according to one of preceding claims, characterized by the following steps: - Separation the chemical sample in a chromatographic separation process by means of the gas chromatography device, - Management of Sample through the connector into the nuclear magnetic resonance spectroscopy apparatus, - Analysis the sample in the detection cell of the nuclear magnetic resonance spectroscopy apparatus. The method of claim 13, wherein the flow of mobile used for the chromatographic separation process Phase is chosen so that the length of stay individual Substances of the sample in the detection cell the time duration for a single nuclear magnetic resonance spectroscopic measurement is needed not below. Method according to one of claims 13 to 14, being the flow of the for the chromatographic separation process used mobile phase between 0.01 ml / min and 50 ml / min, preferably between 0.1 ml / min and 5 ml / min, in particular between 0.1 ml and 1 ml. Method according to one of claims 13 to 15, wherein the sample to a temperature between 0 ° C and 300 ° C, preferably between 10 ° C and 150 ° C, in particular between 20 ° C and 50 ° C he is heated. Method according to one of claims 13 to 16, wherein the separation and the analysis of the sample take place continuously. Method according to one of claims 13 to 17, wherein in the gas chromatography apparatus gas chromatography columns be used, dealing with a lot of sample to be examined load up to 1 mg per substance to be tested. Use of the device and the method according to one of the preceding claims for the separation and analysis of Structural isomers. Use of the device and the method according to one of the preceding claims for the separation and analysis of Stereoisomers.