EP2921237A1 - Centrifuge for automatic sample preparation in a serial workflow - Google Patents

Abstract

The invention relates to a centrifuge machine for automatic sample preparation in a fully automatic analysis system, which cooperates with a robot. This robot has at least one arm for loading and unloading the centrifuge (1, 1 '). The centrifuge machine comprises a centrifuge housing (9) in which a rotor space (11) is defined, in which a rotor (3) for receiving a sample vessel (13) with a substance to be centrifuged and for receiving a reference weight (13 ') arranged and rotatable is stored. The rotor (3) is provided with rocker cups (5, 5 ') for receiving sample vessel (13) and reference weight (13'). For receiving or removing the sample vessel (13) and / or the reference weight (13 '), the rotor (3) having the rocker cups (5, 5') is arranged at a predetermined, defined rotational angular position in the rotor space (11). The invention also relates to a method for centrifuging a sample in an automatic sample processing in a serial process sequence within the fully automatic analysis system.

Description

The present invention relates to a automatic sample preparation centrifuge machine in a fully automatic analysis system cooperating with a robot having at least one arm for loading and unloading the centrifuge, and to a method for centrifuging a sample in such an automatic sample processing in a serial process within one fully automatic analysis system, especially in the field of LC and LC-MS analysis.

Centrifuges are known in various designs. Common to all embodiments is the principle that a rotor having a substance to be separated is rotated in a container by a drive at high speed. As a result, the desired separation of substances located in the rotor takes place. A measure of the speed at which the rotor is rotated in each case is the number of revolutions per unit time. Depending on the type of centrifuge and its intended use, the differences here are very large. So there are centrifuges with speeds in the range of up to 150 000 U / min and those with speeds in the range of several thousand rpm. With the number of revolutions the relative centrifugal acceleration corresponds, abbreviated as RZB or internationally referred to as RCF (relative centrifugal force).

It is also important to distinguish between centrifuges standing on the ground and fixed there, and centrifuges installed on a table. Floor centrifuges are usually operated directly by the rotor having the substances to be separated is mounted on a rotating output shaft of the drive motor. For table centrifuges, it is already known to drive the rotor via a belt drive. Again DE 10 2006 056 134 A1 can be seen, the motor is arranged in this type of drive next to the rotor and not directly connected to this, but the rotational force of the motor is transmitted via a belt to the rotor. For such a belt drive is called the advantage that can be constructed in this way smaller table centrifuges.

As a special feature in the design of the centrifuge rotor so-called swing cup or rocker cups are mentioned. Such swing cups are swingably coupled to the rotor to allow the longitudinal axes of the sample cups carried by the swing cups to pivot outwardly from a vertical orientation in which the sample cups are typically positioned in the rotor outward into a horizontal orientation. when the rotor rotates during the centrifuging process. To balance the dynamic forces during centrifugation, swing bucket / swing cup rotors are typically designed to move the bucket cups substantially symmetrically about the axis of rotation.

By the US 1 769 889 A In this context, a centrifuge arrangement has already become known in the past, which was developed against the background that the price-determining quality of crude oil at the time could only be tested consuming and with loss of time. The revealed there Centrifuge assembly could be operated on a car battery for analysis of crude oil for contained solids.

The centrifuge according to this document has a centrifuge housing with a rotor space. A vertically oriented shaft extending from a motor receives at its end pointing into the rotor space a rotatably mounted transverse arm, which opens in a U-shaped manner in the direction of its two end regions. These U-shaped portions of the transverse arm both serve to receive sample cups, e.g. with crude oil. The legs of the U-shaped opening are bent away in addition to each forming an eyelet down.

The crude oil to be examined is filled into the glass tubes, which are surrounded by a metal container and thus protected. The metal container is inserted into the receptacle on either side of the transverse arm. This photograph refers to the US patent as a "cage", formed of an upper and a lower, diameter slightly smaller ring. The rings are connected by longitudinal struts. In addition, the upper ring still has two opposite and outwardly projecting pins. These pins are used to suspend the "cages" with the sample to be centrifuged on both sides of the transverse arm by hooking them into the eyelets at the ends of the U-shaped legs. When the motor is started, the vertically oriented shaft rotates, with the transverse arm attached to it, and the "cages" with the sample vessels held in them are pivoted outwards.

Recently, there is an increasing demand for efficient analysis systems, e.g. Such an analysis system, with which samples of different types, especially samples from the biological-medical field, such as blood samples, not only examined in the context of a screening, but can be prepared efficiently for this investigation.

This includes that these samples must first be processed method specific, in order to then supply them to the actual meter.

For example, if blood plasma assays are to be performed by LC-MS, the blood plasma must be diluted, an internal standard added, and a precipitant added. The thus treated sample is then shaken and centrifuged to allow the supernatant to be delivered as a measurable sample solution to the LC-MS.

Such preparatory steps required for an analysis, as explained here using the example of the LC-MS analysis, are sometimes cumbersome and time-consuming. They therefore slow the process considerably until a result can be obtained. Methods have hitherto been known which require a number of complex and separate work steps, in particular the method-specific work-up steps already mentioned, which can also result in different time sequences for the individual samples. That The work-up is usually divided into several working steps, which are parallel, i. next to each other and decoupled from the actual measuring system, performed. This results in a large number of possible errors which can falsify the analytical result or make it completely useless.

The centrifuges conventionally used are constructed so that several samples are simultaneously centrifuged and they do not meet the requirements, which requires automatic and serial operation. Since even in the biological-medical field even very small sample quantities with very different matrix have to be worked up and measured, conventional centrifuges can not therefore be used.

The JP 2000-176 317 A discloses a centrifuge designed to avoid imbalance during centrifugation. For this purpose, sample containers are transported via a conveyor into receiving containers, which are located in a centrifuge housing. A rotor is driven by the shaft of a motor and has two arms to which the receptacles for the samples are suspended. Approximately in the middle between you an empty container is hung. The weight of each coming from the conveyor sample container is determined before entering the centrifuge housing on a balance and adjusted with a reference weight. If this results in values which could lead to an imbalance during centrifuging, an alarm is triggered and the delivery of sample containers to the centrifuge housing is stopped.

Based on this, the object of the present invention was to provide a centrifuge with which an automatic sample preparation and analysis in a serial process sequence, in particular in the field of liquid chromatography and especially in the area of coupled LC-MS analysis, i. by further coupling with a mass spectrometer.

This object is achieved by a centrifuge embodied as a vending machine, which is thus suitable for automatic sample preparation in a fully automatic analysis system and interacts with a robot having at least one arm for loading and unloading the centrifuge. To this centrifuge machine includes a centrifuge housing, in which a rotor space is defined, in which a rotor for receiving a sample vessel having at least one substance to be centrifuged, and for receiving a reference weight (13 ') is arranged and rotatably mounted. The rotor has rocker cups for receiving sample vessel and reference weight and the rotor with the rocker cups is arranged for receiving or removing the sample vessel and / or the reference weight at a predetermined defined rotational angular position in the rotor chamber (11).

Depending on the nature of the sample, these steps may include precipitation, liquid-liquid extraction, solid-phase extraction, filtration, derivatization and / or thermal incubation, with small sample quantities for the samples in particular are characteristic, can with the centrifuge according to the invention serially, ie one after the other, centrifuged and fed directly from the sample container to the actual analysis.

So that a serial, ie continuous centrifuging is made possible and this serial centrifuging can be carried out as error-free as possible, the rocker cups for receiving the sample vessel and the reference sample vessel in each case at a defined position on the rotor, arranged in the rotor space.

In this way it can be ensured that the rocker cups can stand or be held in a predetermined, defined (rotational) angular position, so that in this position the sample vessel and / or the reference sample vessel, i. the reference weight, fully automatically positioned in the respective rocker cup or can be removed from this again.

This is done in the centrifuge machine in cooperation with a robot having at least one arm for loading and unloading the centrifuge. In this way, a fully automatic operation of the centrifuge machine is possible.

The centrifuge according to the invention is characterized in a development by a vertical arrangement of the rocker cups of the rotor for receiving the sample vessel and for receiving the reference weight, and up to a horizontal arrangement under the action of centrifugal force in the operation of the centrifuge in the sample vessel and the reference weight , wherein the centrifugal force is effective towards the bottom of the sample vessel.

The sample vessel as well as the reference sample vessel, which has the said reference weight, are thus preferably each positioned vertically in the rocker cups of the rotor provided for this purpose. If, during centrifuging, the centrifugal force acts on the sample in the sample vessel, the centrifugal force in the sample vessel can be swiveled outward by the centrifugal force due to the centrifugal force, so that the centrifugal force in the sample vessel, from top to bottom, towards the bottom of the sample vessel, is effective.

According to a preferred embodiment, the centrifuge according to the invention has an interface to a control unit, so that it is fully automatically controlled and regulated.

Thus, the centrifuge according to the invention is particularly suitable for a serial sample preparation and prepared. The term "serial sample preparation" in the context of the present invention means that one sample after the other is centrifuged in continuous succession and then fed to the analysis. It will not process multiple samples at the same time in the centrifuge.

It is also advantageous for the fully automatic operation of the centrifuge machine when the centrifuge housing accommodating the rotor space has an opening for receiving the sample vessel and the reference weight in the rotor space in its upper region facing away from the bottom of the housing, with a lid for opening and closing the rotor space. is - preferably horizontally - slidably formed, said opening occupies part of the surface of the upper housing portion and the lid remains in the opening in the region of the rotor space.

Because of the rotor space is only partially opened in the automatic mode and must be opened only partially. This is achieved constructively in that the said opening then occupies only a part of the surface of the upper housing portion. In particular, the lid remains in the region of the rotor space during opening. The said constructive measures thus achieve the goal of designing the centrifuge according to the invention as small as possible and space-saving in order to be able to accommodate them at all in a complex, coherent analysis system with a plurality of apparatus subsections.

The centrifuge preferably has a sensor for measuring its acceleration, so that a safety stop can take place in the event of excessive imbalance.

Furthermore, it is preferred that the centrifuge comprises a motor which is connected to a control and regulation unit. As a result, the rotor can be stopped at a defined position and stop.

The centrifuge according to the invention is constructed in such a way that rotational speeds in the range of 2500 to 8000 U / min, preferably 2600 to 7500 U / min are achieved. RPM means number of revolutions per minute.

The invention also relates to a method for centrifuging a sample in an automatic sample processing in a serial process flow within a fully automatic analysis system in which one sample after the other is centrifuged by means of located on a rotor in a rotor chamber of a centrifuge rocking cups for receiving a sample vessel and a reference weight in that the rotor is assigned a sample position and a reference weight position, the rotor is stopped for loading with the sample vessel and / or the reference weight or the unloading at a predetermined defined rotational angular position in the centrifuge and the rotor space for loading or unloading partially opened, wherein the loading or unloading is done with a robot arm.

According to a preferred embodiment, the centrifuge is automatically controlled and regulated via a control unit. It is operated as a centrifuge machine.

According to a development of the method according to the invention, the sample vessel is removed from the rotor space by means of the robot arm and the predetermined part of the centrifuged sample vessel contents is directly fed to an LC analysis, preferably LC-MS analysis.

Direct means that the desired part of the sample is taken from the sample vessel and fed directly to the analysis or its optionally upstream processing steps.

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

Fig. 1:

eine schematische Darstellung einer Analysenvorbereitung mit einer integrierten erfindungsgemäßen Zentrifuge und einer Flüssig-Flüssig-Extraktion zur Vorbereitung einer LC-MS-Analyse, und

Fig. 2:

eine schematische Darstellung einer Analysenvorbereitung mit einer integrierten erfindungsgemäßen Zentrifuge und einer Flüssig-Flüssig-Extraktion kombiniert mit einer Festphasenextraktion zur Vorbereitung einer LC-MS-Analyse, und

Fig. 3:

eine schematische Darstellung des Rotorraums der Zentrifuge mit seinen einzelnen Bestandteilen.

Show it:

Fig. 1:

a schematic representation of an analysis preparation with an integrated centrifuge according to the invention and a liquid-liquid extraction for the preparation of LC-MS analysis, and

Fig. 2:

a schematic representation of an analysis preparation with an integrated centrifuge according to the invention and a liquid-liquid extraction combined with a solid phase extraction for the preparation of an LC-MS analysis, and

3:

a schematic representation of the rotor space of the centrifuge with its individual components.

Specifically, the present invention is designed to constructively alter a centrifuge of known type to be suitable for on-line sample preparation in the sense of serial operation of a fully automated analytical system. This also includes the task of constructing the centrifuge in such a way that it is suitable for use with the stated aim in the field of on-line sample preparation due to a particularly space-saving design.

In order to achieve the stated objects, a centrifuge is used, which will be explained in more detail below with reference to the following Fig. 1 and 2 is provided with the reference numerals 1, 1 '. This centrifuge 1, 1 'is equipped with a known in the art and therefore not shown here, brushless DC motor. It has an attached by means of an adapter on the motor shaft and rotatably mounted rotor plate 3 in the form of an exchangeable rotor disk. Rocker cups 5, 5 ', each for receiving a sample container and a reference sample container, are located on the outside of the rotor cup 3, in order to ensure that the sample container and the reference sample container, as Reference weight is formed, initially perpendicular to the rocker cups 5, 5'hineeingestellt can be.

By the centrifugal force acting during centrifuging they can swing out, the centrifugal force swings the sample container, and according to the reference weight, to the outside.

Under the said rocker cup 5, 5 'is a sleeve to understand, which serves to receive each of the sample container and the reference weight, which are offset by approximately 180 ° to the rotor cup 3.

On the bottom of the rotor cup 3 is further an angle increment. The respective position of the rotor cup 3 is detected in this way, so that defined angular positions and changes can be detected. In addition, a regular speed control is performed.

In the Fig. 1 and Fig. 2 the centrifuge 1, 1 'is shown as embedded as a centrifuge machine within a complete analytical system for analyzing samples 7 from the biological-medical field, which are to be examined in the course of a series investigation with integrated fully automatic sample preparation by means of LC-MS. The samples 7 must first be processed method specific, in order to then be able to perform the actual meter. In the exemplary embodiment, blood plasma examinations have been carried out by means of LC-MS. For this purpose, the blood plasma must be diluted, added to an internal standard and a precipitant added. The thus treated sample 7 is then shaken and centrifuged to allow the supernatant to be delivered as a measurable sample solution to the LC-MS.

While in Fig. 1 Such a sample preparation for a liquid-liquid extraction is shown schematically, relates to the representation in Fig. 2 a sample preparation in which a liquid-liquid extraction and a solid phase extraction (SPE) are possible. The centrifuge 1, 1 'according to the invention can be used equally in both systems. Accordingly, she will be in Fig. 1 with the reference numeral 1 and in Fig. 2 denoted by the reference numeral 1 '. The Centrifuge 1,1 'is therefore also described in total, without between the representations of Fig. 1 and 2 to distinguish or to differentiate.

Accordingly, the centrifuge 1, 1 'on a centrifuge housing 9, in which a rotor chamber 11 is located with the rotor cup 3, to which a rocker cup 5 for receiving a sample vessel 13 with at least one to be analyzed and to be centrifuged first substance and another rocker cup. 5 'are arranged for receiving a reference sample vessel in the form of a reference weight 13'.

Fig. 3 shows once again that the rotor cup 3 laterally the rocker cups 5, 5 ', which are mounted swingably on the rotor cup 3. The sample vessel 13 and the reference weight 13 'are fully automatically positioned, for example, vertically in the respective rocker cup 5, 5' with a dedicated robot technology, which is not shown in the figures. Due to the oscillating cups pivots in the operation of the centrifuge 1,1 'by the then acting centrifugal force both the sample container 13 and the reference weight 13' to the outside and the centrifugal force is from top to bottom, towards the bottom of the sample vessel 13, effective.

A fully automatic sample preparation means that one sample 7 is centrifuged after the other and thus a continuous loading of the centrifuge with the sample vessels 13 takes place. Therefore, the rotor cup 3 requires only a predetermined position for the sample vessel 13 and a position for the reference weight 13 'for computer-implemented control.

Depending on the selected charging function, the rotor cup 3 remains in a defined rotational angle position, so that then the sample vessel 13 and / or the reference weight 13 'can be positioned or removed from the respective oscillating cup 5 via the robot technology.

Since the centrifuge is embedded in a fully automated analysis system, it must be as small as possible, for reasons of space. That the rotor space For this purpose, 11 receiving centrifuge housing 9 has in its upper, the bottom of the housing 9 remote from a (loading) opening 15 for receiving the sample vessel 13 and the reference weight 13 ', which occupies only a portion of the surface of the upper housing portion. For the insertion or removal of the sample vessel 13 and / or the reference weight 13 ', the (charging) opening 15 in the automatic operation is not complete, but only partially, ie partially opened. This is done via a closure lid 17 which is moved horizontally by means of a linear slide 19 for opening and closing. The lid 17 remains in the region of the rotor space 11 during the opening.

In the centrifuge 1,1 'not only the sample vessel 13 and the reference weight 13' fully automatically in the desired manner in the respective rocker cup 5, 5 'positioned, which is possible because the rocker cup 5 for receiving the sample vessel 13 and the rocker cup fifth 'For receiving the reference weight 13' in each case at the already mentioned above, defined predetermined position in the rotor chamber 11 are arranged.

The centrifuge 1, 1 'is altogether controlled and regulated via a data interface defined by an operator software to a computer-implemented control unit. All parameters necessary for centrifuging, which i.a. depend on the type of sample (s) to be centrifuged, determined according to their requirements via the interface protocol. By means of digital inputs and outputs as well as switching contacts, the centrifuge 1, 1 'can thus be easily operated as a whole, as is also necessary for fully automatic operation.

In addition, the centrifuge is 1,1 'sensor controlled. So it has a built-in accelerometer, which ensures that in case of excessive imbalance, typically at +/- 250mg, a safety stop can take place.

• 7500 U/min = ca RCF 4000, entspricht in etwa der maximalen Geschwindigkeit, welche die Zentrifuge gemäß diesem Ausführungsbeispiel erreichen kann
• 6500 U/min = ca RCF 3000
• 4000 U/min = ca RCF 1200
• 2600 U/min = ca RCF 400

By means of a multi-position switch, for the centrifuge 1, 1 'of this exemplary embodiment, specified, specified centrifugal forces can be set, once as revolutions per minute (rpm) and secondly in FIG Form of relative centrifugal acceleration (RZB), internationally known as relative centrifugal force (RCF), are given:

• 7500 rpm = approx RCF 4000, corresponds approximately to the maximum speed, which can reach the centrifuge according to this embodiment
• 6500 rpm = approx RCF 3000
• 4000rpm = approx RCF 1200
• 2600 rpm = approx RCF 400

For the sake of completeness, it should be mentioned at this point that the labeling of the respective sample vessel 13 to be analyzed and of the reference weight 13 'to be analyzed is recorded by image acquisition in order to assign the respective sample to the associated measurement data without any confusion. This is done by the sample vessel 13 is rotated and photographed in a defined angular position. From the images thus obtained, a two-dimensional image is produced, which represents the outside of the sample vessel 13 and also of the reference weight 13 'and can be related to the measurement result for each step within the analysis system.

Claims (10)

Centrifuge machine for automatic sample preparation in a fully automatic analysis system, which cooperates with a robot having at least one arm for loading and unloading the centrifuge (1, 1 '), with a centrifuge housing (9) in which a rotor space (11) is defined in which a rotor (3) for receiving a sample vessel (13), which has at least one substance to be centrifuged, and for receiving a reference weight (13 ') is rotatably mounted, wherein the rotor (3) comprises rocker cups (5, 5 ') for receiving sample vessel (13) and reference weight (13') and wherein the rotor (3) with the rocker cups (5, 5 ') for receiving or removing the sample vessel (13) and / or the reference weight (13'; ) is arranged at a predetermined defined rotational angular position in the rotor space (11). Centrifuge according to claim 1, with a vertical arrangement of the rocker cups (5, 5 ') of the rotor (3) for receiving the sample vessel (13) and for receiving the reference weight (13'), and up to a horizontal arrangement when exposed to the Centrifugal force in the operation of the centrifuge (1, 1 ') in the sample vessel (13) and the reference weight (13'), wherein the centrifugal force in the direction of the bottom of the sample vessel (13) is effective. Centrifuge according to claim 1 or 2, characterized in that it has an interface to a control unit, so that it is fully automatically controlled and regulated. Centrifuge according to Claim 3, characterized in that the centrifuge housing (9) accommodating the rotor space (11) has in its upper region remote from the bottom of the housing (9) an opening (15) for receiving the sample vessel (13) and the reference weight (13 ') in the rotor space (11), with a lid (17) for opening and closing the rotor space (11) which is slidably formed, wherein said opening (15) occupies part of the surface of the upper housing portion (9) and the lid (17) remains open in the region of the rotor space (11) during opening. Centrifuge according to one of claims 1 to 4, characterized in that it comprises a sensor for measuring its acceleration and a safety stop. Centrifuge according to one of claims 1 to 5, characterized in that it comprises a motor which is connected to a control and regulating unit. Centrifuge according to one of claims 1 to 6, characterized by rotational speeds in the range of 2500 to 8000 U / min. Method for centrifuging a sample in an automatic sample processing in a serial process sequence within a fully automatic analysis system, in which one sample after the other by means of on a rotor (3) in a rotor chamber (11) of a centrifuge (1, 1 ') located rocker cups (5 , 5 ') is centrifuged for receiving a sample vessel and a reference weight, the rotor (3) is assigned a sample position and a reference weight position, the rotor (3) for loading with the sample vessel (13) and / or the Reference weight (13 ') or unloading in a predetermined defined rotational angular position in the centrifuge (1, 1') stopped and the rotor space (11) is partially opened for loading or unloading, wherein the loading or unloading is done with a robotic arm. Method according to claim 8, characterized in that the centrifuge (1, 1 ') is automatically controlled and regulated via a control unit. Method according to claim 8 or 9, characterized in that the sample vessel (13) is removed from the rotor space (11) by means of the robot arm and the predetermined part of the centrifuged sample vessel contents is directly fed to an LC analysis, preferably LC-MS analysis.

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