WO2011085791A1

WO2011085791A1 - Method for real-time sample preparation in the hplc high-pressure area with the aid of ultrasound

Info

Publication number: WO2011085791A1
Application number: PCT/EP2010/007823
Authority: WO
Inventors: Werner DÖBELIN
Original assignee: Doebelin Werner
Priority date: 2009-12-21
Filing date: 2010-12-21
Publication date: 2011-07-21
Also published as: EP2521910A1; WO2011085791A9; CH702439A1

Abstract

The invention relates to a high-performance liquid chromatography (HPLC) system, comprising a sample receptacle, an HPLC column (31, 45) in the form of a separating column (45), upstream of which at least one precolumn (31) is arranged, at least one capillary connecting line, and a detector. An ultrasound device (21) is arranged between the sample receptacle and the at least one precolumn (31) and is brought in contact with the or at least one of the capillary connecting line(s). The invention further relates to a method for preparing samples in an HPLC system, wherein the fractions of the sample are finely distributed and homogenized by the ultrasound device (21) or cells in the sample are sufficiently destroyed or the sample is broken down before the sample reaches the at least one precolumn (31). The invention further relates to the use of the HPLC system and method to examine biological, biochemical, pharmaceutical, medical, or environmental samples, in particular for break-down in the protein and molecular range, for protein systems or cell cultures, and for pharmacokinetics or pharmacodynamics.

Description

Method for online sample preparation in high-pressure HPLC using ultrasound

The invention relates to a high performance liquid chromatography (HPLC) system comprising a sample receiver, at least one separation column and a detector, wherein the sample receiver is connected to the at least one separation column by at least one capillary connection line. High-performance liquid chromatography (HPLC) is used as a chromatographic separation method in analytics in a wide variety of fields. These include pharmaceutical research and development, quality assurance, the wide range of life sciences, also known as life sciences, and environmental issues.

Important for the quality of an HPLC test is already the sample preparation or preparation. Great efforts have been made here for a long time to obtain the most accurate and reproducible results with little effort. Especially the processing of biological or medical samples and environmental samples is often associated with great effort, so that the sample for high performance liquid chromatography is suitable and can be injected accordingly into an HPLC system. Sample handling from sampling to sample distribution, sample preparation, sample storage

CONFIRMATION COPY tion to the injection of the sample into the HPLC system harbors a large number of possible errors which falsify the analytical result or even make it unusable. Any simplification of the sample processing minimizes the possibility of error.

In order to ensure that samples to be tested in general and especially biological or environmental samples are suitable for injection into a high-performance liquid chromatograph, conventional processing methods such as centrifuging, precipitation, filtering, extraction, dissolution, derivatization, etc. are known. Significant efforts have been made, particularly in the field of filtration, to provide sample preparation filtration systems for a large volume bandwidth. In addition to the large number of possible volumes that can be investigated, a distinction must be made between single and multiple samples as well as automated systems with regard to sample preparation. In particular, it is known to work with syringe filters which had to be redesigned especially for automated HPLC systems. Here are multilayer prefilter have become known, which are equipped with, inter alia, glass fibers in order to ensure a higher throughput, coupled with faster flow rates. Such multilayer filters hold particles in the order of> 40 to 1 μπι back.

In the field of extraction, it is known to use solvent-compatible filter media in the form of membranes. Here is the problem of artifacts due to extractable components of the filters. In addition, depending on the type, the membranes can interact with the sample to be investigated by means of electrostatic, ionic, covalent, hydrogen or other binding, which can likewise lead to falsifications.

Although centrifugation sample preparation is basically considered to be a fast, ie time-saving and simple method, it is not free from disadvantages. Centrifugation involves centrifugal forces of up to 14,000 g. Here it is important to prevent leakage through special sealing methods. Since, for this type of sample preparation, filter In addition, care must be taken to prevent the entry of undesirable, extractable constituents.

With the measures mentioned, e.g. sufficient preparation for such samples is guaranteed, which clump or tend to clump. These can still clog the HPLC column. For blood samples with intact red blood cells, the normal analytical HPLC columns clog due to the size of these blood cells. In addition, molecules are generally to name, which preferably adhere to each other and thereby often poorly separated by chromatographic means.

On this basis, the invention therefore an object of the invention to provide a system for sample treatment, with which these negative effects can be reduced with reasonable effort and new applications can be made possible, so that in particular biological, biochemical, pharmaceutical, medical and / or environmental samples directly after the Sampling, optionally after intermediate storage, injected into an HPLC system and can be analyzed with high quality. This object is achieved according to the invention with a high-performance liquid chromatography (HPLC) system which has a sample holder, at least one HPLC column and a detector, wherein the sample holder is connected to the at least one HPLC column by at least one capillary connection line. In the region between the sample holder and the at least one HPLC column, an ultrasonic device is arranged and brought into contact with or with at least one of the capillary connecting lines.

The term HPLC column is to be understood quite generally and includes any type of columns which can be used and used in an HPLC system. First, in the simplest case, the HPLC column is to be understood as a separation column. The HPLC system can be operated in normal phase or reverse phase. ben. The reverse phase method, the most common HPLC method, is commonly referred to as "reversed phase" and abbreviated to RP.

It is also customary to connect one or more precolumns to the actual separation column. On the one hand, these have the task of preventing impurities from the separating column as the main column and, on the other hand, they can serve for concentrating. In the context of the teachings of the invention shown here is initially not distinguished between precolumn and separation column. If the ultrasound device is arranged in the region between the sample holder and the at least one HPLC column, it may be arranged either in front of the separation column or in front of the first pre-column, depending on whether and how many pre-columns are provided.

Since it is the task of the ultrasound device to produce as homogenized a sample as possible which passes through the at least one HPLC column without clogging it, or else to destroy cells in the sample in order to access internal cell components for analysis it makes sense according to the invention to arrange the ultrasonic device in front of the respective first HPLC column, ie before the separation column or before the first guard column.

According to the invention, the ultrasonic energy acts directly on and into the sample. This is to homogenize the sample, the components contained in the sample are crushed and the property (s) of the sample changed so far that a clogging of the at least one HPLC column is avoided. In principle, this achieves a substantial improvement and simplification of the analysis.

When the sample after the high-pressure injection is pumped through the at least one connecting capillary which is brought into contact with the ultrasonic device, the connecting capillary should be rigidly connected to the ultrasonic head of the ultrasonic device so that the ultrasonic vibration is as complete as possible the connection capillary is transferred and the ultrasonic energy can thus act directly and as completely as possible on the sample. As a result, the sample is homogenized and optimized for the at least one HPLC column. The HPLC column does not clog or significantly less. The service life of the HPLC column (s) is extended and the separation efficiency of the HPLC column can improve.

In an HPLC system which, in addition to a separation column, also has one or more precolumns upstream of the separation column as HPLC columns, the ultrasonic device is arranged in the region between the sample receiver and the at least one guard column. In the presence of a plurality of guard columns, the ultrasonic device is in principle also arranged in front of the first guard column. Only in this way can it fully unfold the effect that it has achieved in accordance with the invention.

According to a further embodiment of the HPLC system according to the invention, the at least one capillary connecting line can have essentially three sections or consist of at least three capillaries, which are each connected to one another. Usually, such compounds are achieved by capillaries in the HPLC system via so-called fittings, which are known per se. The first section of the capillary connection line or the first capillary then leads from the sample receiver to the ultrasonic device. The second portion or capillary is brought into contact with the ultrasonic device and the third portion or capillary is connected to the at least one HPLC column which, when only one HPLC column is present in the HPLC system, is the separation column is. If several HPLC columns are present, it is a precolumn. According to a further embodiment, the ultrasound device has an ultrasound head and the second section of the capillary connection line or the second capillary is brought into contact with the ultrasound device by being wound onto the ultrasound head. The two further sections of the capillary connecting line or the two further capillaries are, on the other hand, kept flexible.

In this way, the part of the capillary connection line, which is directly connected to the ultrasound head by being wound up, can absorb sound energy well and pass it on to the inside of the capillary. However, no ultrasonic energy is lost since the two parts of the capillary leading respectively to the ultrasound device are kept flexible. As a result, the ultrasonic vibration is damped in these areas and not forwarded.

The material for the capillary (s) may be selected from metal, which may additionally be coated with glass fibers in a variant in Kapillarinnern and / or plastic. In this case, the metal may be selected from stainless steel, stainless steel or titanium and / or the plastic may be selected from polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), in particular Teflon, and / or fluoropolymers, in particular ethylene-tetrafluoroethylene (ETFE). The capillaries to be used must always be suitable for high pressure. Preferred Kapillarstärken therefore arise for the skilled person, depending on the material used from the requirement to withstand the respective pressure of the system. As preferred capillary thicknesses, the outside diameter may be about 1/32 "to 1/8" and the inside diameter is 0.25 to 2 mm, preferably 1/16 "as the outside diameter, and 0.25 to 0.75 mm, particularly preferably 0.5 mm as the inside diameter.

The invention also relates to a method for sample preparation in an HPLC system, in which the sample is injected via a sample receptacle in the HPLC system and passed over at least one capillary connection line to at least one HPLC column. In this case, the sample is brought into contact with an ultrasonic device by which their proportions are finely divided and homogenized before the sample reaches the at least one HPLC column. Again, the term HPLC column is to be understood to include both the separation column and the precolumn, depending on whether the HPLC system has one or more columns, at least one of which regularly precolumn. Conventional ultrasonic frequencies of the ultrasound device are in the range of approximately 10 to 60 kHz. According to the invention 20 to 50 kHz, more preferably about 30 kHz are used. In order to ensure sufficient homogenization of the sample, if appropriate, sufficient cell destruction and / or digestion of the sample, not only the frequency but also the amplitude must be taken into account. The ultrasound energy or the energy flow is controlled by the amplitude. As a further measure, the residence time of the sample in the ultrasonic range, ie to call the effective path length, which depends inter alia on the flow rate and the residence time of the capillary in direct contact with the ultrasonic head. In addition, ultrasound can be applied as continuous sound or pulsed. According to the invention, pulsed ultrasound with a pulse width of 10-100% is preferably used. According to the invention, it may be provided that after the ultrasonic device, the sample first passes at least one HPLC column in the form of a precolumn, which is connected upstream of the further HPLC column in the form of a separation column. This includes the case that more than one guard column is connected upstream. Within the scope of the present invention, it is particularly preferred if the sample intake in the HPLC system takes place directly from a sample taken from a body or medium or from a sample supply.

Then, the advantage of the invention ultrasonic comminution can be exploited particularly effective and time-saving. An elaborate sample preparation, as required in the prior art, can be completely eliminated.

A body or cell culture or other sample from or from which the direct sampling is made may be, for example, that of a small animal or child. Then comes another advantage to bear, which can be further enhanced by the use of a specially designed pipette tip, as it has become known with WO-A-2010/084180. This advantage consists in the small amounts of sample needed to perform the HPLC analysis. On the one hand, this is due to the fact that the usual Preparation and preparation steps for the sample require additional sample volume, which is not necessary here. On the other hand, the sample volume can still be removed from the body or medium in a particularly gentle manner and in the smallest amounts by the said pipette tip in such a way that the pipette tip itself can already be used for injection into the HPLC system.

The invention also relates to the use of the HPLC system as described above in various embodiments and the corresponding method for sample preparation in the HPLC system for the examination of biological, biochemical, pharmaceutical, medical and / or environmental samples.

Here it is especially to do with disturbing influences of the sample, which often can not be eliminated by a pretreatment. This includes, in particular, the analysis of blood samples with intact red blood cells. But even without the background of interfering influences, the HPLC system according to the invention can e.g. for digestion processes in the protein and molecular range, for protein systems or cell cultures as well as for pharmacokinetics and / or pharmacodynamics.

In the following the invention will be explained in more detail with reference to an embodiment and the accompanying drawings.

Show it:

1 shows a schematically illustrated HPLC system with ultrasonic device and pre-column circuit with a guard column,

2 shows a schematic representation of the ultrasonic device,

and Fig. 3: a schematically illustrated HPLC system with ultrasonic device and pre-column circuit with three pre-columns.

To illustrate the sample preparation according to the invention in a high-performance liquid chromatography (HPLC) system with the aid of ultrasound, FIG. 1 shows an HPLC system which can operate both in normal mode and in reversed-phase mode. The ultrasound treatment of a sample 1 to be examined is completely independent thereof and will be explained in more detail below.

In addition, the following statements apply not only for use in the field of conventional HPLC separation methods. The inventive design of the HPLC system, i. Substantially sample preparation before passing through the column (s) has proven to be usable and suitable in the field of ultrahigh-performance liquid chromatography (UPLC or UHPLC) as well. The UPLC differs from the conventional HPLC separation process by a higher sample throughput, which is essentially conditioned by a higher separation efficiency achieved in the UPLC process. Basically, the UPLC refers to a HPLC separation process with greatly enhanced performance. This definition is also used in the context of these remarks.

The HPLC system shown in FIG. 1 has a sample receiving area into which a sample 1, which has at least one analyte to be examined, is transported directly from a sample container 3. The sample container 3 has in this embodiment, a special embodiment, which is particularly advantageous, but not necessarily required to carry out the present invention. Therefore, this special design of the sample container 3 will be discussed in more detail below.

In the sample receiving area is an input line 5, which is connected to a valve 7, so that the sample 1 with appropriate circuit of the Valve 7 passes from the sample container 3 in a sample loop 9. An overflow line 11 ensures that a sample overflow can flow into a designated waste container 13. By switching the valve 7, the injection of the sample 1 into the pre-column area of the HPLC system is then carried out. For this purpose, an HPLC charge pump 15 is provided, through which the sample 1 located in the sample loop 9 is guided via a line 17 and with appropriate switching of the valve 7 into a first line section 19 of a capillary connection line and from there to the sample preparation according to the invention for an ultrasonic device 21 arrives.

This ultrasonic device 21 is shown in Fig. 2 again in more detail. It has an ultrasound driver 23, an ultrasound adapter 25 and an ultrasound head 27. The direction of movement of the ultrasound thus generated extends axially on the axis designated by the reference numeral 28 in FIG. On the ultrasonic head 27, a second line section 29 of the connecting line is wound and coupled by this winding substantially rigidly, but releasably coupled to the ultrasonic head 27. This ensures that the ultrasonic energy acts directly on the sample 1, which is guided over the first line section 19 in this second line section 29, due to the turns of the second line section 29 dwells in the region of the ultrasonic head 27 and this happens several times. The number of windings of the second line section 29 is thus also a measure of the residence time of the sample 1 in the ultrasonic range. The windings are chosen so that the sample 1 is well homogenized by the ultrasonic treatment, i. solid, agglomerated or clumped components in the sample are sufficiently destroyed by the ultrasound effect. The constituents of the sample 1 are finely distributed in this way and optimized for an HPLC precolumn designated by the reference numeral 31 in the exemplary embodiment. It has been found that the HPLC pre-column 31 is not or significantly less clogged than without ultrasound treatment. The lifetime of the HPLC pre-column 31 is thus considerably extended.

However, the use of the HPLC system according to the invention is not limited to this field of application. For digestions in protein and molecular rich, in protein systems or cell cultures, the cells or molecules are burst and unlocked. This has opened up new investigation methods, especially in the field of pharmacokinetics and pharmacodynamics. Thus, the HPLC system investigations were carried out in the field of efficacy testing of active pharmaceutical ingredients, which were previously not possible and which are basically based on destroying cells suitable for the purpose of the investigation. This method is used in the field of pharmacokinetics or pharmacodynamics to test for certain active ingredients in cells and to determine how much of them contained in the cells to be examined, that is, actually penetrated into the cells. Here, the study is based on a cell culture in the form of a concentrated cell matrix, which is about 10 times smaller than whole blood. This cell matrix is pipetted, injected into the HPLC system, the cells are ruptured, and the drug released thereby can be determined. By the prior preparation of the concentrated cell matrix can now be ensured beyond doubt that the specific concentrations of active substance were actually in the cell, or were relatively firmly bound to the outer cell wall, so they do not by the previous steps of concentration and washing could be replaced.

The ultrasonic device used for the above-described experiments had used an ultrasonic frequency of 30 kHz. The ultrasound energy or energy flow was controlled by the amplitude. In addition, pulsed ultrasound was used with a pulse width that was varied from 10-100% for testing purposes. Another important factor was the residence time of the sample in the ultrasonic range, i. the effective path length the sample traveled in the ultrasound range per unit of time. Here, the flow rate for the residence time of the capillary is in direct contact with the ultrasound probe and the capillary itself, i. their inside diameters play a role.

In the test experiments flow rates were set, which allowed a residence time of the samples depending on the experiment carried out from 5 to 30 s. Especially while winding numbers of the capillary 29 were tested around the ultrasonic head 27 of 25 - 35 windings. In particular, the experiments were adjusted so that at a winding number of 30 and a flow rate of 100 - 300 μΐ per min, a residence time of about 20 s in the ultrasonic range resulted.

After passing the ultrasonic device 21, i. essentially the ultrasonic head 27, the connecting line passes into a third line section 33, through which the sample 1 is passed after passing through a valve 35 via a further line 37 to the already mentioned HP LC-Vorsäule 31.

First line section 19, second line section 29, in which the sample 1 is brought into contact with the ultrasound by passing the windings around the ultrasonic head 27, and the third line section 33 are integrally formed in the embodiment.

With regard to the capillaries to be used, it should be noted in principle that they must be suitable for high pressure. The preferred capillary material has been found in the context of the test trials PEEK. Other plastic materials and capillaries of steel or titanium can also be used.

Thin-walled, high-pressure PEEK capillaries with an outer diameter of about 1/16 "and an inner diameter of 0.5 mm were mostly used here.

Depending on the application further capillary thicknesses with an outer diameter of 1/32 "and 1/8" and an inner diameter of 0.25 - 0.75 mm or larger can be used.

In this case, the first line section 19 and the third line section 33 are flexibly mounted and held to thereby dampen the ultrasonic vibration and not forward.

The at least one analyte in the sample 1 is now retained by the HPLC pre-column 31. Further, non-retained components of the sample 1 are further rinsed by the HPLC charge pump 15 via output lines 39 and 41 into a second waste container 43. In this way, the at least one analyte in the sample 1 on the HPLC pre-column 31 is cleaned for the first time, concentrated on it. trated and separated from interfering components. The at least one analyte in the sample 1, which is now still on the HPLC pre-column 31, is now forwarded to an analytical separation column 45. For this purpose, a HPLC pump 53 is actuated via a valve 47 and lines 49 and 41, and the sample 1 flows via lines 55 and 57 to the analytical separation column 45 where it is again separated from interfering components which are in the sample 1 to then pass via a line 59 in a specific detector not shown in the figures.

In the context of this embodiment, an MS-MS detector was used. The use of MS-MS coupling by a triple-quad device makes it possible to obtain as much information as possible from the samples to be examined. By this method fragments of molecules could be measured. Alternatively, any HPLC detectors may be used. In order to test the described HPLC system more closely, biological samples from the area of blood, plasma, urine and cerebrospinal fluid were preferably used and injected directly after sampling. The HPLC system was also coupled to a mass spectrometer. This is considered in the context of the experiment explanation only insofar as a successful analysis of the samples with the mass spectrometer, after the chromatographic separation via HPLC, in the direct injection of these biological samples in the first place was possible and surprisingly successful, not by artifacts to false peaks or led in other way falsified measurement results.

It was also considered in the investigations that the samples examined, especially blood samples taken from mice, were only available in a very small amount for the respective analysis.

It should again be pointed out that direct injection of the samples under investigation in the context of the present invention is understood to mean, in particular, an injection which involves the conventional preparatory steps in the examination of a sample, such as metering into a sample loop. should be removed from a sample vessel and the washing steps required thereby avoid the parts in contact with the samples. Also, no prior solid phase extraction or other usual preparation step is required.

For direct injection, a sample container, not shown in more detail in the drawing, is used, which will be discussed in more detail. This sample container, as disclosed by WO-A-2010/084180 /, to which reference is made in its entirety, has a pipette tip which has an open end both on the pipette side and on the sample side. Inside the pipette tip is an inner support sleeve provided with a capillary opening. The sample-side end of the pipette tip is pressure-tight pressed into a front side sealed with a membrane closure sleeve. The closure sleeve with the pipette tip located in it is clamped in a holding device, wherein holding jaws are used for holding the closure sleeve and the pipette tip. On a feed system insertable capillary needles are arranged axially in the pipette tip. In this way, sufficient small amounts of sample can be obtained per se for an analysis and injected directly into the HPLC device according to the invention. Ultrasonic treatment avoids sample loss. The term "smallest sample amounts" are to be understood as meaning amounts of 1-50 μΐ, preferably of 5-10 μΐ, and are sufficient for the analysis.

When injecting sample 1 by means of this sample container, the pipette tip closes the sample loop of the HPLC injection system. The sample loop is replaceable and the sample container in which sample 1 is prepared and injected with the pipette tip is disposable, i.e., it may be discarded after injection.

For automated measurement, prepared boxes containing 54 sample containers filled with the above-mentioned pipettes can be used. They can either be stored or used directly for a measurement. The pipettes are described in the above mentioned WO-A-2010/084180. ben. The thus prepared for the measurement sample container is closed on one side by a closure sleeve. On the other side is the support sleeve.

In the experimental examples described below, the actual separating column 45 was preceded by one to three pre-columns 37. The criteria according to which one, two or three pre-columns 37 have been used according to the invention apply in general and in particular also for all HPLC investigations explained below and shall be briefly presented below in general terms. The use of one or more guard columns is not necessarily required, but has proven to be useful to keep impurities as possible of the actual separation column 45 as the main pillar. The number depends on the system to be examined. A single-stage pre-column circuit 37 was chosen when the sample to be tested could be directly injected into the HPLC system without prior sample preparation. This basically depends on the nature of the analytes to be investigated. In such a single-stage pre-column circuit 37, the sample to be tested is injected directly into the HPLC system without prior sample preparation steps and the described pipette tip represents a subsequently disposable sample loop. Using the set of e.g. 54 pipettes, the measurement can be fully automatic. Then the pipettes or pipette tips are replaced automatically after each injection. The tests used a pressure of 2,500 psi, corresponding to 170 bar. Higher pressures are possible in principle.

Moreover, if the pre-column circuit 37 has been explained with the ultrasonic module 21 according to the invention as a single-stage pre-column circuit 37, this is due to the fact that it depends essentially on the ultrasound module that for the purposes of the present invention easiest on the basis of the single-stage Vorsäulenschaltung 37 can be explained. This is therefore not to be understood as limiting. For those skilled in the art, oh- ne further that the principles described for the ultrasonic module 21 according to the invention can also be used when using a two-stage and multi-stage Vorsäulenschaltung. A two-stage precolumn circuit, as shown in FIG. 3, has proven to be expedient if a solid-phase extraction step via an SPE (solid phase extraction, SPE) column in conjunction with a RAM precolumn 31 'proved to be expedient , This is the case, for example, with (blood) serum and plasma samples. The pipette or pipette tip already described is used again as a subsequently disposable sample loop. With fully automatic measurement, the set of 54 pipettes serves as an injection system. The pipettes or pipette tips are automatically replaced as described above after each injection. In the tests, a pressure of 2,500 psi, corresponding to 170 bar, was applied again, with higher pressures being possible in principle. This two-stage pre-column circuit was used in two versions, once in the usual HPLC with the pressures already mentioned for the one-stage pre-column circuit, and in the UPLC version with a working pressure of up to 20,000 psi. The UPLC will be explained in more detail below.

A two-stage pre-column circuit may therefore be indicated when a two-phase cleaning step is required, e.g. once via an SPE precolumn and additionally on a RAM column. In the present case, RAM columns are understood as meaning columns of "restricted access materials" based on silica gel with hydrophobic groups, and combined with the reversed-phase mode of the HPLC system, it was thus possible to disrupt molecules by determining the sizes of fractions of molecules ,

Use of said RAM columns in the presently investigated samples was considered necessary in particular because they were preferably biological samples.

RAM columns are suitable for low molecular weight hydrophobic analytes from non-pretreated samples, especially biological samples, which extract and enrich directly. Such biological samples include hemolyzed blood, plasma, serum, milk, fermentation broth, supernatants or food homogenates. An example of their application is the study carried out in the context of the present invention for the determination of bosentan and three of its metabolites directly from the blood in the form of an HPLC-MS assay. The blood samples were purified on a RAM column, commercially available from Merck in Darmstadt, Germany, concentrated on a precolumn and separated on a C-phenyl HPLC column HPLC flow rate on said analytical separation column 45 was 300 μΐ / min in the exemplary embodiment. Basically much smaller, but also higher flow rates are just as possible. The detector used was an MS-MS detector. In the test runs under the HPLC / UPLC studies were 45 particles having a particle diameter of 2 for the separation column - used 5 ^μ η ι as column material. As a result, both the speed and the performance, ie the separation performance of the column 45 could be improved. The higher working pressure required for this variant could be made possible in particular by using the previously described pipettes for the HPLC or UPLC injection system by using suitable pumps and valves. With regard to the valves, high pressure valves designed for a working pressure of up to 20,000 psi were used.

In particular, it has been shown that the analysis time in the context of the UPLC separation process by the combination of the aforementioned pipettes, or pipette tips, in combination with the ultrasonic device according to the invention could be shortened particularly favorable. The special pipette system provides a fast, direct sampler. However, full development of its advantages does not occur until the ultrasound device according to the invention is coupled with this direct sampler. The ultrasonic device makes a direct sample application without prior preparation of the sample in the first place possible and thus meaningful.

Claims

claims

High-performance liquid chromatography (HPLC) system, with a Probenaufhahme, at least one HPLC column (31, 45) and a detector, wherein the Probenaufhahme with the at least one HPLC column (31, 45) is connected by at least one capillary connecting line, characterized in that an ultrasound device is arranged in the region between the sample receiver and the at least one HPLC column (31, 45) and brought into contact with or with at least one of the capillary connecting lines.

HPLC system according to claim 1, characterized in that the HPLC column (31, 45) is a separation column (45) preceded by at least one precolumn (31) and in that the ultrasonic device (21) is located in the region between the sample receptacle and the at least one guard column (31) is arranged.

HPLC system according to claim 1 or 2, characterized in that the at least one capillary connecting line has substantially three sections or that the capillary connecting line consists of at least three capillaries which are respectively connected to each other, of which the first section (19) or the first capillary from the sample holder to the ultrasonic device (21), the second section (29) or the second capillary is brought into contact with the ultrasonic device (21), and the third section (33) or the third capillary with the at least one HPLC Column (31, 45) or the separation column (45) is connected.

HPLC system according to claim 3, characterized in that the ultrasonic device (21) comprises an ultrasound head (27), that the second section (29) of the capillary connection line or the second capillary by winding on the ultrasound head (27) in contact with the ultrasonic device is brought, and that the two other sections of the capillary connecting line (19, 33) or the other two capillaries are kept flexible.

HPLC system according to one of Claims 1 to 4, characterized by a material for the capillary (s), which is selected from metal, in the capillary inside with glass fibers coated metal and / or plastic.

HPLC system according to claim 5, characterized in that the metal is selected from stainless steel, stainless steel or titanium, and / or that the plastic is selected from polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), in particular Teflon, and / or Fluoropolymers, especially ethylene tetrafluoroethylene (ETFE).

Method for sample preparation in an HPLC system, in which the sample is injected into the HPLC system via a sample receiver and conducted via at least one capillary connection line to at least one HPLC column (31, 45), characterized in that the sample with an ultrasonic device (21) is brought into contact, by which their shares finely divided and homogenized and / or cells in the sample are sufficiently destroyed and / or the sample is digested before the at least one HPLC column (31, 45) reached.

8. The method according to claim 7, characterized in that a pulsed ultrasonic wave having a frequency between 10 and 60 kHz, preferably 20 to 50 kHz, particularly preferably 30 kHz is used.

9. The method according to claim 7 or 8, characterized in that the sample is held in the at least one capillary connecting line for about 5 to 30 s in contact with the ultrasound. 10. The method according to any one of claims 7 to 9, characterized in that the sample after the ultrasonic device (21) initially at least one HPLC column (31, 45) in the form of a precolumn (31) passes, the further HPLC column ( 31, 45) in the form of a separation column (45) is connected upstream.

10. The method according to any one of claims 7 to 9, characterized in that the sample is taken in the HPLC system directly from a sampling of a body or medium or from a sample supply.

11. Use of an HPLC system according to any one of claims 1 to 6 or the method for sample preparation according to one of claims 7 to 10 for the investigation of biological, biochemical, pharmaceutical, medical and / or environmental samples.

12. Use according to claim 11 for the analysis of blood samples with intact red blood cells, for the digestion in the protein and molecular range, for protein systems or cell cultures as well as for pharmacokinetics and / or pharmacodynamics.