Apparatus and method for accurate pipetting independent of liquid class - Patent application

Abstract

The present application relates to a pipetting device (10) and a pipetting method for pipetting, i.e. aspirating and / or dispensing, a metered liquid (32) using a working gas, independently of the flow or / and wetting properties of the metered liquid (32), wherein the pipetting tube (12) is maintained at its known reference temperature (T ∞ ) is located in a lower reference temperature region, and its known operating temperature (T AB2 and a second operating region (AB2) in which the first operating region (AB1) is located in an operating temperature region higher than the reference temperature region.

Description

[Technical field]

[0001] The present invention relates to a pipetting device for pipetting, i.e. for aspirating and / or dispensing, a metered amount of liquid with a working gas, which pipetting device comprises: a pipette tube extending along a tube axis, a pipette piston movable within the pipette tube along the tube axis; a receiving space for receiving a metering liquid, the receiving space extending in the pipette tube along the tube axis from the pipette opening at one end to a metering piston face of the pipette piston facing the pipette opening at the other end, a working gas being received in the pipette tube directly in contact with the metering piston face, the reference volume of the working gas being determined by the volume of the working gas present in the receiving space under a reference pressure of the working gas, a drive connected to the pipette piston in a force-transmitting manner, the drive being configured to displace the pipette piston along the tube axis; a position detection device for detecting the position of the pipette piston along the tube axis and outputting a position detection signal representative of the detected position; a pressure detection device for detecting the pressure of the working gas in the pipette tube and outputting a pressure detection signal representative of the detected pressure; and - a control device, Contains The control device controls the drive device. -Pressure detection signal, - reference pressure of the working gas, and - the determined target metered liquid volume to be pipetted, The control device is configured to operate taking into account the pressure change induced change in the reference volume of the working gas brought about by the piston movement according to the method of the present invention, and the control device is further configured to perform the piston movement required for pipetting the target metered liquid volume in a number of successive movement steps. [Background technology]

[0002] The present invention further relates to a method for accurately pipetting a metered amount of liquid using a pipetting device.

[0003] The pipetting device and the pipetting method according to the present application are exclusively concerned with the pipetting process of the so-called "air displacement" method, in which between the metering liquid to be pipetted and the piston face of the pipette piston facing the metering liquid there is a trapped amount of working gas having a working gas volume, the value of which is at least in the range of the order of magnitude of the volume of the metering liquid to be received in the pipette tube. In general, the volume of working gas trapped between the metering piston face and the metering liquid is larger than the volume of the metering liquid received in the pipette tube.

[0004] In this case, in the present application, a pipette tip, possibly connected to a pipetting device, is considered to be part of the pipette tube. Generally, the metering liquid is only aspirated into such a pipette tip and dispensed from the pipette tip, more precisely from a container aspirated into the pipette tip. Furthermore, in this case, the pipette tip is generally not completely filled with the metering liquid. The volume of the enclosed working gas amount is generally between 50 μl and 1000 μl. This applies equally to the prior art and to the present invention.

[0005] The accuracy of the pipetting process in terms of the amount of metered liquid received or dispensed depends on the properties of the metered liquid, such as its viscosity, density, wetting behavior with respect to the material of the pipette tube, and surface tension. If different liquids with the above mentioned different properties are pipetted with the same pipetting device with the same pipetting parameters, such as the displacement path and displacement speed of the piston, this will generally result in different amounts of metered liquid dispensed or aspirated for both liquids, depending on in which direction the pipetting process proceeds.

[0006] Up to now, in pipetting technology, this situation has been taken into account by dividing the metered liquid into classes of liquids with the same or sufficiently similar pipetting properties. For each of the liquid classes thus formed, correction values ​​may be stored in the data storage device of the pipetting device, which correction values ​​are applied to the pipetting parameters in order to displace the pipette piston in such a way that the actual volume of the pipetted metered liquid coincides as accurately as possible with the target volume to be pipetted. For example, for liquids with a large flow resistance, particularly those with high viscosity, the volume to which the pipette piston must advance may be larger by a certain factor than the metered liquid volume to be pipetted by the pipette piston movement, and / or the displacement speed of the pipette piston during pipetting may be reduced from a reference value in order to compensate for the large flow resistance and to pipette the desired metered liquid amount as accurately as possible.

[0007] It is disadvantageous that the liquid class of the liquid to be pipetted must be known in order to be able to pipette the liquid as accurately as possible with the air displacement method. In fact, for a large number of metering liquids, there are liquid classes and correction values ​​associated with said classes. However, significant difficulties arise when a metering liquid of an unknown class is to be pipetted, for example when a metering liquid consisting of a mixture of various liquids and not assigned to one liquid class is to be pipetted. In this case, through complex experiments, the unknown metering liquid must be assigned to a liquid class or new liquid classes must be set and defined in order to be able to pipette the unknown metering liquid accurately.

[0008] A pipetting device of the initially mentioned kind is known from DE 10 200 03 133 A1. DE 10 200 03 133 A1 discloses a pipetting device in which the pressure changes in the working gas volume trapped in the pipetting tube, which are brought about by the movement of the pipetting piston, are taken into account, so that the working gas volume trapped in the pipetting tube can be determined as accurately as possible after the end of the pipetting piston movement, from which the volume of the metering liquid present in the pipetting tube can be deduced. In this case, according to its explanation, DE 10 200 03 133 A1 assumes the ideal gas equation. However, in practice, the compensation of the volume change of the working gas induced by the pressure change taught in DE 10 200 03 133 A1 is based on a special case of the Boyle-Marriott law, since DE 10 200 03 133 A1 assumes a purely isothermal state change of the working gas.

[0009] Additionally, the pipetting method known from EP 1 099 633 A1 considers a purely empirical residual amount of metering liquid, which flows off, possibly due to inertia, after the end of the piston movement for pipetting through the pipette opening of the pipette tube, and EP 1 099 633 A1 does not state the exact cause of the residual amount that flows off later, but merely points out the empirical circumstances for determining the residual amount.

[0010] From EP 1 099 436 A1 a pipetting device is known in which the pipette tube has two differently temperature-regulated regions, i.e. a region located closer to the pipette piston with a higher working gas temperature and a region located closer to the pipette opening with a lower working gas temperature. Thus, when the pipette piston moves, the working gas moves from one region to the other and is heated or cooled depending on the direction of movement, which again results in a volumetric change of the heated or cooled amount of working gas. EP 1 099 436 A1 teaches to correct the target volume to be pipetted by the temperature-induced volumetric change of the working gas and to pipette a correspondingly increased or decreased target volume.

[0011] The temperature compensation of the target pipetting volume taught in US Pat. No. 5,399,633 is performed according to Gay-Lussac's law, which assumes isobaric changes of state, i.e. constant pressure of the heated or cooled working gas.

[0012] In addition, from US Pat. No. 5,399,543 a method and a device are known for contactlessly determining the volume of material received in a closed container, for which the value of exactly one state variable of a gas contained in the closed container is changed, namely the pressure, volume, temperature or amount, and from the resulting change in the remaining state variables of said gas the gas volume and ultimately the material volume are deduced using the ideal gas equation. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] European Patent No. 1250956 [Patent Document 2] U.S. Pat. No. 5,895,838 [Patent Document 3] DE 19651252 A1 Summary of the Invention [Problem to be solved by the invention]

[0014] Based on what has been described so far, the object of the present invention is to describe a technical teaching that allows accurate pipetting of a metered liquid without predetermining the liquid class of the metered liquid and without predetermining the physical properties of the metered liquid that are relevant for pipetting, such as viscosity, density, wettability, surface tension, etc. [Means for solving the problem]

[0015] According to a first aspect of the invention, the problem is solved by a pipetting device of the type mentioned at the beginning, the pipetting tube of which has a first operating area, the known reference temperature of which is located in a lower reference temperature area, and a second operating area, the known operating temperature of which is located in an operating temperature area higher than the reference temperature area, the control device being adapted to determine, after a first pipette piston movement step, for a subsequent pipette piston movement step: determining a first correction variable representative of a pressure change-induced volume change of a portion of the working gas volume trapped in the pipette tube, the portion being located in a first operating region, based on the position detection signal, the pressure detection signal and a reference pressure of the working gas; determining a second correction variable representative of a pressure-change- and temperature-change-induced volume change of a portion of the working gas volume trapped in the pipette tube located in a second operating region based on the position detection signal, the pressure detection signal, a reference pressure of the working gas, the known operating temperature, and the known reference temperature; The target step movement volume of the pipette piston is - the step reference volume to be assigned to the subsequent piston movement step, - the displacement volume of the metering piston surface of the pipette piston so far during the ongoing pipetting process, a first correction variable, and - the second correcting variable, and actuating the drive device in accordance with the determined target step motion volume.

[0016] The working gas reference pressure is preferably the pressure of the working gas at the start of the pipetting process. If the pipette tube before the aspiration process is filled exclusively with working gas, the working gas reference pressure is preferably the ambient pressure of the pipetting device, since the working gas in the pipette tube is in pressure-compensating contact with the gas of the surrounding atmosphere through the pipette opening. If at the start of the aspiration process a certain amount of metering liquid has already been received in the pipette tube, the working gas reference pressure can again be the ambient pressure or the working gas pressure at the start of the aspiration process. The same applies to the dispensing process. At the start of the dispensing process a certain amount of metering liquid is always received in the pipette tube, which is dispensed through the pipette opening. In this case too, the working gas reference pressure can be the working gas pressure at the start of the dispensing process or the ambient pressure.

[0017] In this application, a "pipetting process" or an "ongoing pipetting process" refers to a pipetting process in which the receipt or ejection of a target metered volume of liquid is assigned as a pipetting result.

[0018] The reference volume of the working gas may be the dead volume of the pipette tube between the pipette opening and the metering piston surface, which is filled exclusively with working gas, at a defined position of the pipette piston in the pipette tube, for example in a reference state in which the pipette piston is at its bottom dead center, the pipette tube is filled exclusively with working gas and the pipette opening is open, thereby allowing pressure communication between the working gas and the environment outside the pipetting device. The dead volume may be physically separated from the environment by immersing the pipette opening in a metering liquid container. When the pipette opening is placed on the liquid level of the metering liquid container, the ambient pressure prevails in the working gas in the pipette tube, which is isolated from the environment.

[0019] The target metered liquid volume is typically set as a target value for the pipetting process and is therefore known.

[0020] The step reference volume for a pipette piston movement step can be set independently of the target metering liquid volume to be pipette, i.e. it can be predetermined, for example taking into account the structure and kinematics of the pipetting device.

[0021] Alternatively, the control device can determine the step reference volume as a reference degree of movement of the metering side piston surface for the following pipette piston movement step, given the target metering liquid volume. This can be done, for example, by using a calculation formula stored in the data storage device, which calculates the step reference volume for one or more pipette piston movement steps according to a predefined number of pipette piston movement steps for the execution of a pipetting process for pipetting the target metering liquid volume and according to the target metering liquid volume to be pipetted during the pipetting process. This can also be done alternatively by reading out the step reference volume from a predefined data correlation, which assigns a step reference volume to each pipette piston movement step based on the number of pipette piston movement steps and the target metering liquid volume as raw data.

[0022] The step reference volume may be a set of values ​​clearly determined for a predefined number of pipette piston movement steps. Alternatively, the step reference volume may be, for example, a quotient of a target metered liquid volume and a predefined number of pipette piston movement steps. The predefined number of pipette piston movement steps may be the total number of pipette piston movement steps of a pipetting process if the target metered liquid volume should be distributed evenly among all pipette piston movement steps.

[0023] Preferably, the number of pipette piston movement steps in which the target metered liquid volume is dispensed is smaller than the total number of pipette piston movement steps of the pipetting process, so that the pipetting process additionally includes a pipette piston movement step in which the piston moves in a movement volume smaller than the pipette piston movement step of the target metered liquid volume, not to receive a larger amount of metered liquid, but predominantly or exclusively to compensate for the metered liquid volume already received in the pipette tube. However, the value of the step reference volume is preferably different for each pipette piston movement step of the pipetting process, so that knowledge and experience gained so far about pipetting liquid can be taken into account. For example, during pipetting, especially during aspiration, the value of the incremental step reference volume for a pipette piston movement step closer to the start of the pipetting process is preferably larger than the value for a pipette piston movement step closer to the end of the pipetting process. In this way, the overflow behavior or / and backflow behavior of the metered liquid during pipetting, especially during aspiration, can be taken into account.

[0024] The step reference volume, which is predetermined or can be determined as described above, is used as the reference step size of the pipette piston along the tube trajectory for each subsequent pipette piston movement step. The step reference volume adapted to the target metered liquid volume and the target step volume assigned to the same pipette piston movement step will not differ in value more than the step reference volume and the assigned target step volume of the same pipette piston movement step, which is predetermined independently of the target metered liquid volume. However, this is not of any importance for the success of pipetting, since it is of no fundamental importance which part of the determined target step volume of a pipette piston movement step is based on the step reference volume and which part is based on the step correction volume, which supplements the step reference volume to the target step volume.

[0025] The liquid class independent open or closed loop control of the pipetting process discussed in this application focuses on the working gas, not the metering liquid, because of the working gas in the pipette tube interacting with the pipette piston, and is therefore preferably based on the ideal gas equation, which predicts the behavior of the working gas very well, so in this application the movement distance of the pipette piston is expressed as a volume. The volume is directly processed in the ideal gas equation. Based on the size of the metering side piston surface known through the respective configuration, the step volume can be easily converted into the pipette piston step size along the tube trajectory, and the pipette piston can be actuated for the corresponding movement.

[0026] The control device may be an electronic data processing device, for example including at least one integrated circuit. Preferably, the control device has a data storage device, in which an operating program and operating data are stored, and the electronic data processing device outputs a control command to the driving device based on the operating program and the operating data.

[0027] The working gas may be any working gas, in many applications it will simply be air, but it may also be a noble gas, e.g. helium or argon, or a semi-inert gas, e.g. nitrogen or carbon dioxide, if required, for example by the metering liquid to be treated.

[0028] The displacement volume of the metering piston surface up to a subsequent pipette piston movement step in the ongoing pipetting process is the volume displaced by the metering piston surface through a displacement along the tube trajectory in the ongoing pipetting process. The displacement volume takes into account the pipetting operations performed up to now in the pipetting process. The displacement volume is therefore a measure for the pipetting operations performed up to a subsequent pipette piston movement step in the ongoing pipetting process.

[0029] With the previous movement of the pipette piston, the amount of working gas present in the pipette tube has been manipulated. The working gas has been displaced and / or its volume has been changed, i.e. for example expanded during aspiration and compressed during dispensing. The above-mentioned correction variables, the first correction variable and the second correction variable, can be used to determine as accurately as possible the effect of the previous pipette piston movement in the pipetting process on the working gas. It is then possible to determine as accurately as possible the amount of metering liquid present in the pipette tube through the manipulated working gas.

[0030] By performing the pipette piston movement stepwise and determining a target step movement volume for each subsequent pipette piston movement step using the above-mentioned parameters, a target metered liquid volume to be pipette in a pipetting process can be pipette with high precision.

[0031] For the sake of simplicity, the pipette tube is divided into two operating regions. Basically, it is not excluded that further operating regions are determined, but two operating regions are already sufficient for high-precision pipetting results. The operating regions differ according to the temperature prevailing therein, where the temperature of the working gas is important. The working gas in the first operating region is in a lower reference temperature region, and for the first operating region, the first correction variable only considers the volume change induced by the pressure change. The working gas in the second operating region is in a higher operating temperature region, and for the second operating region, the second correction variable considers the volume change based on the pressure and temperature changes of the working gas.

[0032] The pressure change as a cause of the volume change is brought about by the previous movement of the pipette piston. Due to said movement of the pipette piston since the beginning of the pipetting process, the pressure of the working gas has changed from the initial working gas pressure, in particular the reference working gas pressure, to the current working gas pressure. This pressure change again causes a change in the amount of metering liquid in the pipette tube, because besides other physical effects, such as friction, the pressure of the working gas prevailing in the pipette tube is important for a certain amount of metering liquid to be retained in the pipette tube. The difference between the current working gas pressure and the working gas pressure, in particular the reference working gas pressure, at the beginning of the pipetting process is therefore a measure for the change in the amount of metering liquid received in the pipette tube. But also, with the change in the working gas pressure during the pipetting process, the volume of the working gas trapped in the pipette tube has changed from the initial working gas volume to the changed working gas volume. This volume change is taken into account for each subsequent pipette piston movement step when determining the amount of metered liquid present in the pipette piston after the preceding pipette piston movement step.

[0033] The first working area is preferably an area under relatively constant external conditions. The first working area is preferably located in an ambient atmosphere, for example having a constant or approximately constant ambient temperature. The first working area preferably includes the pipette tip, which is releasably connectable with the rest of the pipetting device, and the part of the pipette tube fixed to the device, which is possibly exposed to the ambient atmosphere. It is not too far-fetched to assume that these parts are permanently at the constant temperature level of the ambient atmosphere.

[0034] The second operating area of ​​the pipette tube may be a portion of the pipette tube that is not directly exposed to the ambient atmosphere and / or whose temperature is not decisively affected by the ambient atmosphere, unlike the first operating area, and for the second operating area, not only are the volume changes of the working gas caused by the pressure changes due to the piston movement corrected, but also the volume changes of the working gas caused by the temperature changes between the reference temperature and the operating temperature are corrected.

[0035] By determining the correction value and applying it before each pipette piston movement step, on the one hand, the value of the correction carried out during each movement step is smaller than if the correction was determined once for the entire pipetting process, and on the other hand, by the correction of the step reference volume to the target step movement volume before each pipette piston movement step, a pipetting with high precision in terms of the pipette amount is obtained.

[0036] If in the present application it is stated that the position detection device detects the position of the pipette piston along the tube axis, this includes both direct detection of the pipette piston position, insofar as the detection result represents the pipette piston position, and also indirect detection of the pipette piston position, since the goal is achieved that the position detection device can output a position detection signal representative of the position of the pipette piston. Correspondingly, mutatis mutandis, applies to detection of the pressure of the working gas in the pipette tube by a pressure detection device. The working gas pressure can also be detected directly or indirectly, insofar as the detection result represents the working gas pressure in the pipette tube.

[0037] The invention starts from the observation of the pipette tube volume between the pipette opening and the metering piston face. The entire volume between the pipette opening and the metering piston face is filled with working gas or with working gas and metering liquid. In the following, the invention will be described using the example of an aspiration process, which is more difficult to control with high precision than a dispensing process. In addition, the initial state of a dispensing process is generally formed by aspiration, so that the aspiration process can be considered to be at the beginning of each pipetting process.

[0038] At the beginning of the aspiration process, a known position of the metering piston surface between the pipette opening and the metering piston surface in the pipette tube, i.e., a position equivalent to a known position of the pipette piston, is occupied exclusively by the working gas dead volume V T The pipette opening is slightly immersed in an external measuring liquid container, which reduces the dead volume V T is isolated from the external environment, but the metering liquid does not enter the pipette tube through the pipette opening by capillary action, the dead volume of the working gas isolated from the surroundings V T It is useful to use as the reference volume of the working gas. The pressure of the working gas at that time is the reference pressure of the working gas, and therefore the ambient pressure p ∞ The temperature of the working gas is the ambient temperature T ∞ It is.

[0039] First, in order to explain the basic principle behind the present invention, the division of the pipette tube into two differently temperature-regulated working regions should be ignored. The amount of working gas trapped between the metering liquid and the metering piston face remains constant for the further pipetting process, if we ignore the evaporation processes in the pipette tube and the leakage processes from the pipette tube. In contrast, the volume of the trapped working gas does not remain constant.

[0040] When immersed, the metering piston surface has a displacement volume V KolbenWhen the pipette moves away from the pipette opening by a volume V, the volume of the dispensed liquid increases based on the negative pressure thus created relative to the initially prevailing ambient pressure. liquid flows from the container through the pipette opening into the pipette tube. The volume of the received metered liquid V liquid It is very important for an accurate aspiration process to know the working gas pressure p1, which is different from the reference working gas pressure, at the working gas temperature T1, which is different from the initial temperature, in the pipette tube. That is, the initial working gas dead volume V T First, V Kolben Then, the volume of the metered liquid that flows in V liquid Therefore, the working gas volume V that exists in the pipette tube after the pipette piston movement is l is obtained as follows:

[0041] equation 1

[0042]

number

[0043] ideal gas equation

[0044]

number

[0045] or

[0046]

number

[0047] Using the above initial conditions we get:

[0048] equation 2

[0049]

number

[0050] Using equations 1 and 2, we can determine the volume V in terms of the unknown variables. liquid You get:

[0051] equation 3

[0052]

number

[0053] That is, the movement of the pipette piston creates a volume V Kolben The amount of liquid received by V is liquid is a known V T This can be calculated at p ∞ If p1 and p2 can be detected by a pressure detection device, T ∞ and T1 is detectable by a temperature detection device, or T ∞ and T1 are known, and V Kolben is detectable by a position detection device.

[0054] However, the invention is complicated by the differently temperature-regulated working areas of the pipette tube. Typically, the first working area is located near the pipette opening, while the second working area is located near the metering piston surface. According to a preferred further development of the invention, the first working area starts at the pipette opening and runs from the pipette opening into the pipette tube, while the second working area is located along the heat source of the pipetting device. If the metering piston surface is in the second working area, the first and second working areas preferably adjoin each other and abut between the pipette opening and the metering piston surface, depending on the construction of the pipetting device. The working gas volume V1 enclosed in the above-mentioned pipette tube is the volume fraction of the first working area. AB1 V1 and the volume of the second operating region AB2 It is composed of V1 and V2.

[0055] equation 4

[0056]

number

[0057] Essentially, in this application, the value assigned to the first operating region is designated "AB1" and the value assigned to the second operating region is designated "AB2."

[0058] Initial working gas dead volume V T extends into both operating regions, equation 4 already accounts for the initial working gas dead volume V T (see equation 4' below):

[0059] Equation 4'

[0060]

number

[0061] Alternatively, the working gas volume trapped between the metering liquid and the metering piston surface can be displaced only with the pipette piston movement during the pipetting process, such that the working gas volume extends into both working regions after the displacement process. T extends initially only in one active area, this active area is generally the first active area.

[0062] Preferably, for simplicity, the first operating region is constant, e.g., at ambient temperature T ∞ However, the assumption is made that the temperature is at a first temperature level represented by T AB1 However, for the reasons mentioned above, it makes sense to assume that the ambient temperature is a constant temperature in the first operating area. Therefore, the aspirated metering liquid is as far as possible not heated or, particularly preferably, does not change temperature in the pipette tube, since the metering liquid is generally only aspirated in the first operating area.

[0063] If we assume the preferred case that the metering piston surface is located at or near its bottom dead center at the start of the above-described exemplary aspiration process, then the largest possible piston stroke is available for the reception of the metering liquid, and the volume V displaced by the pipette piston during the aspiration process or during a partial step of the aspiration process is Kolben can be located in the first operating area or / and in the second operating area. Thus, quite generally, the following is valid:

[0064] equation 5

[0065]

number

[0066] AB1 V Kolben may be 0 and the entire piston motion is within the second operating region. AB1 V Kolben If is not 0, the first AB2 V T = 0 must be valid because the initial working gas dead volume V T This is because the piston movement causes the working gas to be displaced along the tube trajectory only in the first operating region, but no displacement of the working gas occurs between the first and second operating regions.

[0067] That is, it may be assumed that during the piston movement, the working gas is isothermally expanded or compressed in the first operating region. This means that the part of the volume displaced by the metering piston surface located in the first operating region AB1 V Kolben also applies.

[0068] As long as the entire volume of working gas trapped between the metering liquid and the metering piston face during the pipetting process is only in the first operating region, open or closed loop control of the pipetting process is not an issue, since changes in the working gas are considered and treated as isothermal state changes. The invention comes into play when the metering piston face moves or starts to move into the second operating region.

[0069] In contrast, the volume of the working gas corresponding to the volume part located in the second operating region of the piston movement AB2 V Kolben is displaced between the first and second operating regions. During aspiration, displacement occurs from the first operating region to the second operating region, and during dispensing, displacement occurs in the opposite direction. In the second operating region, the temperature of the working gas T AB2 is T AB2 >T ∞ With the displacement of the working gas between the first and second operating regions, a temperature change of the working gas is effected.

[0070] During isothermal and isobaric piston movement, the volume in the pipette tube displaced by the metering piston face will correspond to the change in the amount of metering liquid in the pipette tube, since wherever there is no working gas in the pipette tube, there must be metering liquid.

[0071] However, piston movement cannot result in an isobaric state change of the working gas, since only a pressure change in the working gas can do work on the metering liquid and displace it through the pipette orifice.

[0072] Because the working gas is displaced between a first operating region and a second operating region of higher temperature, the change in state of the working gas caused by the piston movement does not have to be isothermal.

[0073] Based on the above assumptions, the part of the piston movement located in the second operating region is the working gas volumeAB2 V Kolben is displaced between the first and second operating regions. This displaces the working gas volume AB2 V Kolben The temperature of the ∞ From T AB2 When dispensing, AB2 From T ∞ Additionally, the working gas volume is subject to pressure changes caused by the piston movement.

[0074] The part located in the first operating area of ​​the piston movement is the working gas volume AB1 V Kolben This only brings about a change in pressure. At this time, the working gas volume V, which corresponds to the total volume displaced by the metering piston surface, Kolben changes based on pressure and temperature changes by the following values:

[0075] equation 6

[0076]

number

[0077] A first operating area extending from the pipette opening of the pipette tube through the structural shape of the pipette opening creates a volume V AB1 The volume V of the metered liquid received in the pipette tube caused by the piston movement liquid is the volume displaced by the metering piston surface V Kolben In the first operating region, under the simplifying assumption that the piston surface of the metering side corresponds to the system-specific residual volume of the working gas when the piston surface of the metering side is in the second operating region, AB1 V sys,rest remains, for which the following formula is valid:

[0078] equation 7

[0079]

number

[0080] V T is the initial volume in the second operating region when the metering piston surface is initially, i.e., at the beginning of the pipetting process, in the second operating region. AB2 V init For that much, V AB1 It can be larger than V T V AB1 If the piston surface is smaller than 1 mm, the piston surface on the metering side first moves to the boundary between the first and second operating regions, AB1 V Kolben Therefore, equation 7 is most commonly expressed in terms of equation 5 as:

[0081] equation 7 *

[0082]

number

[0083] AB2 V init or AB1 V Kolben Only may be different from 0, because AB1 V Kolben is always subtracted from the equation, AB1 V Kolben Because it is not important.

[0084] Preferably, V AB1 ≧V T And without any logical contradiction AB2 V init If =0 is valid, for example, the first operating region starts at the pipette opening and passes the bottom dead center or other starting position of the metering piston surface, so that Equation 7 * can be simplified and written as:

[0085] Equation 7'

[0086]

number

[0087] Since the system-specific residual volume, by definition, exists only in the first operating region, the subscript "AB1" is omitted below when describing the system-specific residual volume. Therefore, the system-specific residual volume V sys,rest extends in the described operating state of the pipetting device from the edge of the pipette tube or from the meniscus of the metering liquid facing the working gas in the pipette tube to the boundary of the first operating area remote from the pipette opening, so that the system-specific residual volume is the volume displaced by the metering piston surface during the piston movement, possibly located in the first operating area. AB1 V Kolben The residual volume V sys,rest According to the above assumptions, the system undergoes an isothermal state change due to the piston motion, which results in a specific residual volume V sys,rest is the following value ΔV sys,rest Change by:

[0088] equation 8

[0089]

number

[0090] equation 7 * and equation 7' ​​directly shows that the volume displaced by the metering piston surface in the first operating region is already taken into account in the system-specific residual volume. Taking into account the part of the piston movement located in the first operating region and the changes in the system-specific residual volume induced exclusively by the pressure change, and furthermore, since both operating regions can be in pressure-compensating communication with each other, the same working gas pressure p AB2 =p AB1From equation 6, only the part of the piston motion that lies in the second operating region remains, under the valid assumption that θ exists. This leads from equation 6 and equation 8 to the following equation 9:

[0091] equation 9

[0092]

number

[0093] The volume of the metered liquid received in the pipette tube differs from the volume of the piston movement that results in the reception of the metered liquid in the pipette tube by the volume changes of equations 8 and 9. Expressed by the formula, this means that:

[0094] equation 10

[0095]

number

[0096] Using Equation 6, Equation 7, and Equation 8, Equation 9 can also be expressed as:

[0097] Equation 10'

[0098]

number

[0099] In another grouping, Equation 10' gives rise to the following Equation 10":

[0100] Equation 10”

[0101]

number

[0102] Volume V Tis a purely structural dimension of the pipetting device and is therefore known. The temperature T AB2 Knowing the volume V displaced by the metering piston surface, which can be detected by the position detection device, Kolben and a portion of the second operating region. AB2 V Kolben and the pressure p of the working gas in the pipette tube, which can be detected by the pressure detection device. AB1 Knowing the initial parameters p ∞ , T ∞ Knowing φ, it is possible to approximately calculate the volume of metered liquid received in the pipette tube for each pipette piston movement step using one of Equation 10″, Equation 10′ or Equation 10, independent of the pipetting characteristics of the metered liquid.

[0103] Temperature quotient

[0104]

number

[0105] is, by definition, always positive and greater than 1.

[0106]

number

[0107] is likewise always positive and regularly greater than 1, since the working gas pressure in the pipette tube cannot be negative and must in most cases be less than the ambient pressure in order to be able to hold the metering liquid received in the pipette tube in the pipette tube. Exceptions can only occur when the volume received in the pipette tube of a particular metering liquid is small, in which case the metering liquid can be held in the pipette tube by capillary forces. In this case, p AB1 is the ambient pressure p ∞ It can be bigger than that.

[0108] In Equation 10, the term ΔV sys,rest is an example of a first correcting variable in the sense of the present application, and the term AB2 ΔV Kolben is the second correcting variable in the sense of the present application.

[0109] In equation 10', the term

[0110]

number

[0111] is an example of a first correcting variable in the sense of the present application, and the term

[0112]

number

[0113] is an example of a second correcting variable in the sense of the present application.

[0114] In equation 10, the term

[0115]

number

[0116] is an example of a first correcting variable in the sense of the present application, and the term

[0117]

number

[0118] is an example of a second correcting variable in the sense of the present application.

[0119] In Equation 10, Equation 10' and Equation 10", V Kolbenis the magnitude of the displacement volume of the metering piston face so far in the ongoing pipetting process. Equation 10, Equation 10' and Equation 10" all relate to the same state of the pipetting device, V liquid yields the same value of

[0120] At the end of the pipetting process, V liquid represents the desired metered volume of liquid very accurately. For an aspiration process starting from the reference state of the pipetting device defined above, V liquid is the pipetted metered liquid volume that corresponds very precisely to the target metered liquid volume at the end of the pipetting process. Start V liquid For a pipetting process starting from 0.5°C, the pipetted metered liquid volume, which corresponds very precisely to the target metered liquid volume, is determined at the end of the pipetting process by the difference between the initial and final volume of the metered liquid in the pipette tube, i.e. V liquid - Start V liquid However, the "pre-filled" operating state is likewise derived from the above-mentioned description of the aspiration process, since the initial state of such a "pre-filled" pipette tube must itself return again to the aspiration process, in which the pipette tube is initially filled only with working gas. The same applies to the dispensing process, the initial state of which must also have been previously aspirated.

[0121] As already indicated, the control device may be configured to determine an estimate for the amount of metered liquid present in the receiving space based on the displacement volume of the metering side piston surface of the pipette piston in the ongoing pipetting process, the first correction variable and the second correction variable for a subsequent pipette piston movement step, in order to obtain particularly accurate pipetting results. Such an estimate may be determined, for example, using at least one of the above-mentioned equations 10, 10' and 10". liquid - Start V liquidis an estimate that makes it possible to determine the volume of the pipette metered liquid in the pipette tube as the volume after each pipette piston movement step. Start V liquid For V = 0, i.e., when aspirating the metered liquid starting from the reference state described above, liquid It is an estimate itself.

[0122] The control device may further be configured to compare the thus determined estimate with the step reference volume for a subsequent pipette piston movement step and determine a target step volume based on the comparison result. For example, the target step volume may be determined according to a difference value between the estimate and the step reference volume. In this case, a larger difference value preferably results in a larger target step volume than a smaller difference value.

[0123] In this case, the step volume is preferably used as the cumulative step volume, which represents the sum of all the individual step volumes that the pipette piston has advanced in the previous movement steps. In this case, according to the above explanation, the cumulative step volume after the end of the last movement step of the pipetting process preferably corresponds to the target metered liquid volume, referred to as a volume. The comparison of the estimated value and the cumulative step volume therefore corresponds to a comparison of the target metered liquid volume assigned to the movement steps performed so far, represented by the cumulative step volume, with the actual metered liquid volume actually pipetted, represented by the estimated value.

[0124] The differential value may be converted by the control device into a step-corrected motion volume through a proportional or / and differential or / and integral conversion element. The proportional conversion element determines the value of the proportional step-corrected motion volume based on the differential value and with a proportionality factor to be determined by experiment or experience. The differential conversion element determines the value of the differential step-corrected motion volume based on the difference between the current differential value and the previous differential value, i.e., based on the change in the differential value, with a differential weighting factor to be determined by experiment or experience. The integral conversion element determines the value of the integral step-corrected motion volume based on the sum of the differential values, including the current differential value, with an integral weighting factor to be determined by experiment or experience.

[0125] The target step motion volume of the subsequent pipette piston motion step may be the step reference volume assigned to the subsequent pipette piston motion step corrected by the step correction motion volume.

[0126] The control device is preferably configured to carry out successive pipette piston movement steps until the difference value between the specific estimated value and the step reference volume falls below a predefined difference threshold, in particular for a predefined number of directly successive pipette piston movement steps. In this case, the target metered liquid volume is generally pipetted with a pipetting accuracy determined by the difference threshold. This interruption criterion provides, in particular, under additional conditions, a highly accurate pipetting result in which the step reference volume is the cumulative step reference volume and the pipetting process has progressed to such an extent that the cumulative step reference volume corresponds to the target metered liquid volume.

[0127] Alternatively, the control device may be configured to perform a predefined number of directly successive pipette piston movement steps, which is sufficient to ensure accurate pipetting of viscous metering liquids with high surface tensions as well, so that for less viscous metering liquids, no appreciable pipette piston movement is performed any more before the predefined number of pipette piston movement steps is reached, since the estimate already corresponds approximately to the target metering liquid volume very early on.

[0128] To obtain highly accurate pipetting results, the control device may be configured to perform more than 100, preferably more than 1000, particularly preferably more than 10000 pipette piston movement steps per second. Based on the dynamic limitations of the pipette piston, the control device is configured to perform less than 100000 pipette piston movement steps per second.

[0129] The control device can read out the step reference volume assigned to a subsequent pipette piston movement step from the data storage device according to the target metered liquid volume and / or calculate it based on the target metered liquid volume, as already described in detail.

[0130] The step reference volume may be an incremental step reference volume, which represents, starting from the current position of the metering side piston surface, for one subsequent pipette piston movement step, the step reference volume to which the metering side piston surface should advance during this next movement step. Advantageously, the cumulative value of the step reference volume through an increasing number of steps, obtained by summation from the previous incremental step reference volumes, has a first step region, during which the cumulative value increases from an initial value to a value of at least 95%, preferably exactly 100%, of the target metered liquid volume, and a second step region following the first step region, during which the cumulative value does not leave the range of 95% to 105%, preferably exactly 100%, of the target metered liquid volume.

[0131] The step reference volume may alternatively be an absolute step reference volume, also denoted above as cumulative step reference volume, which represents the final position of the metering side piston face starting from the starting position of the metering side piston face at the beginning of the pipetting process. Again, the value of the absolute or cumulative step reference volume has a first step region in which the value of the absolute step reference volume increases from an initial value through an increasing number of steps to a value of at least 95%, preferably exactly 100% of the target metered liquid volume, and a second step region following the first step region in which the value of the absolute step reference volume does not leave the range of 95% to 105%, preferably exactly 100% of the target metered liquid volume.

[0132] Advantageously, the proportion of the second step area in the overall pipetting process is greater than 20%, preferably greater than 30%, in terms of the number of pipette piston movement steps, so that towards the end of the pipetting process there is a deceleration phase during which the pipette piston mainly performs correction movements, which increases the accuracy of the match between the target metered liquid volume and the actually pipetted metered liquid volume. The pipette piston movement steps performed in this phase generally have a smaller target step movement volume than in the first step area, which allows an undisturbed and sufficiently stable control of the pipetting process. Thus, in the second step area, the formation of mutual disturbances during the pipetting process and their corrections can be largely eliminated due to the smaller target step movement volume. For the same reason, the second step area preferably has at least the same number of steps as the first step area, or a larger number of steps, and / or preferably lasts for at least the same time length as the first step area, or for a longer time length.

[0133] In case of uncertainty, the start of the pipetting process should coincide with the start of the first step region, i.e. at a time when the initial value of the incremental step reference volume is not zero. In the pipetting device according to the invention and in the pipetting method according to the invention, even though the step reference volume further only has the value of zero from the time of immersion of the pipette opening in the metering liquid container, a movement of the pipette piston can occur, for example, in order to resist the obstacle of the metering liquid passing through the pipette opening, which is brought about only by capillary forces. However, such a piston movement is only a correction movement that can last for any length. In case of uncertainty, the pipetting process according to the invention starts when the pipette piston starts to move, by which the incremental step reference volume changes from the initial zero to a value different from zero. In this case, the difference between two cumulative or absolute step reference volumes that are directly successive in time is equal to the incremental step reference volume.

[0134] For a control method that is as stable as possible, with only slight possibility of being influenced by external factors, the control device calculates the determined target step movement volume based on the following three parameters: - the target final position of the metering piston surface at the end of the next pipette piston movement step, - the target displacement speed of the piston face during the subsequent pipette piston movement step, and - the time length of the subsequent pipette piston movement step, The method may be configured to determine the above by quantifying at least two of the following parameters:

[0135] Thus, not only the position of the metering end face but also the displacement speed can be defined. For example, in the data storage device of the above-mentioned control device, different displacement speeds or movement durations can be stored for different values ​​of the target metering liquid volume and / or for different values ​​of the step reference volume.

[0136] As already shown with Equation 10, Equation 10' and Equation 10'', the control device may be configured to form a second correction variable based on the portion of the working gas located in the second operating region and a product of a pressure quotient and a temperature quotient, where the pressure quotient is the quotient of the detected working gas pressure and the working gas reference pressure, and the temperature quotient is the quotient of the operating temperature and the reference temperature.

[0137] Likewise, for a high-precision and high-speed movement of the metering piston surface, it is advantageous if the drive comprises a linear motor, the rotor of which is the pipette piston. In this case, the drive preferably comprises a number of energizable coils, which are arranged radially outside the pipette tube along the drive section in order to provide a sufficiently high driving force to the pipette piston. In order to obtain a driving force on the pipette piston as uniform as possible, the coils preferably close-wrap the pipette tube in the circumferential direction around the tube axis. Since the at least temporarily energized coils form a heat source, the second operating area comprises the drive section. The volume displaced by the metering piston surface during the displacement of the metering piston surface is therefore preferably completely within the second operating area. For reasons of thermal conditions that are as well predictable as possible, the first operating area extends along the tube trajectory up to the arrangement area of ​​the coils. Thus, preferably, the part of the pipette tube extending from the pipette opening-side longitudinal end of the arrangement area of ​​the coils to the pipette opening is exposed to the external environment of the pipetting device and forms the first operating area.

[0138] Essentially, in dynamic thermal equilibrium, a known operating temperature can arise during the operation of the pipetting device in the second operating area. This equilibrium operating temperature can be stored in the above mentioned data storage device and can be used to control the pipetting process. However, detection of the temperature by a sensor in the second operating area is more reliable and accurate than reliance on a constantly re-occurring state of thermal equilibrium. Therefore, the pipetting device according to a preferred further development of the invention comprises a temperature sensor for detecting the operating temperature, which outputs an operating temperature signal representative of the operating temperature.

[0139] To avoid contamination by successive pipetting of different metering liquids with the same pipette tube, the pipette tube preferably has a tube part fixed to the device with a connecting structure and a pipette tip releasably connected to the connecting structure, the pipette tip having a pipette opening, and the metering liquid is pipetted only into the pipette tip and not into the tube part fixed to the device.

[0140] In addition to pressure- and temperature-induced changes in the reference volume of the working gas during the pipetting process, unavoidable leakage in the area surrounding the working gas can also adversely affect the pipetting accuracy. The working gas can, for example, flow past the pipette piston or over the connection structure for connection of the pipette tip. Therefore, preferably, the control device is configured to determine a third correction variable representing the leakage of the pipette tube, and the control device is configured to additionally determine the target step movement volume of the pipette piston based on the third correction variable. In this case, for example, the above-mentioned equation 10 can be modified as follows:

[0141] Equation 11

[0142]

number

[0143] In this case, preferably, the leakage volume ΔVLeckage The third correction variable is preferably the structure-dependent leakage rate K L and the pressure inside the pipette tube, p AB1 and ambient pressure p ∞ It depends on the pressure difference between the

[0144] equation 12

[0145]

number

[0146] In order to quantify the leakage volume as a third correction variable, the control device may be configured to determine the third correction variable based on the pressure detection signal and the time length, in particular the time length of the pipette piston movement step.

[0147] The control device further comprises a third correction variable, L Since the leak parameter may change over time for a given pipetting device, the controller may be further configured to determine the leak parameter based on manual control input or by predetermined automatic control to perform detection of the change over time of the initially determined working gas pressure affected by the leak in order to update the value of the leak parameter.

[0148] For example, the control device can, based on a manual control input or by a predetermined automatic control, for example at regular predetermined time intervals, connect a pipette tip with a closed pipette opening or without a pipette opening to a tube section fixed to the device, adjust a predetermined pressure of the working gas in the pipette tube through the movement of the pipette piston, and detect the change in the working gas pressure over a predetermined time period. From the change in the working gas pressure over the predetermined time period and from the pressure difference between the working gas pressure in the pipette tube and the ambient pressure of the pipetting device, the control device can quantify a leakage parameter.

[0149] The pipetting device may have a pressure sensor for detecting the ambient pressure. Alternatively, the ambient pressure may be manually input through an input device.

[0150] The invention also solves the problem set out at the beginning by a method for accurately metering a metering liquid independently of the flow properties or / and wetting properties of the metering liquid by means of a pipetting device, in particular by means of a pipetting device with further developments as described above, the pipetting tube having a first operating region, the known reference temperature of which lies in a lower reference temperature region and a second operating region, the known operating temperature of which lies in an operating temperature region higher than the reference temperature region, the method performing a stepwise displacement of a pipette piston movably accommodated in the pipette tube, the method comprising, after a first movement step of the pipette piston, for subsequent pipette piston movement steps, the following method steps: - detecting the pressure of the working gas; - detecting the position of the pipette piston; - determining a first correction variable representative of a pressure change induced volume change of a first portion located in a first region of a volume of working gas enclosed in a pipette tube based on the detected pipette piston position, the detected working gas pressure and a reference working gas pressure; determining a second correction variable representative of a pressure- and temperature-induced volume change of a portion of the working gas volume trapped in the pipette tube, the portion being located in a second operating region, based on the detected pipette piston position, the detected working gas pressure, the working gas reference pressure, the known operating temperature and the known reference temperature; determining an estimate for the metered liquid present in the receiving space based on the detected pipette piston position, the previous pipette piston position, a first correction variable and a second correction variable; - determining or retrieving from a data storage device a step reference volume assigned to a subsequent pipette piston movement step; - comparing the estimated value with the step reference volume; - determining a target step movement volume for a displacement of the pipette piston in a subsequent pipette piston movement step; and Displace the pipette piston by the target step motion volume.

[0151] The operating temperature may be known because it occurs in the second operating area as an equilibrium temperature during operation of the pipetting device, but the method may also comprise the step of detecting the operating temperature in the second operating area of ​​the pipetting tube.

[0152] Further developments of the above-mentioned pipetting device, which preferably operates according to the method, are also further developments of the method according to the invention and vice versa.

[0153] The invention will now be described in detail with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0154] [Figure 1] 1 shows an embodiment of a pipetting device according to the present application at the start of an aspiration method according to the present application as a pipetting method according to the present application; [Diagram 2] 2 shows the pipetting device according to FIG. 1 after a first, relatively small, suction has progressed during a pipetting method; FIG. [Diagram 3] FIG. 3 shows the pipetting device according to FIGS. 1 and 2 after a second, relatively large, suction step has progressed during the pipetting method. [Figure 4A] FIG. 2 shows a graph of the aspiration process according to the present invention, together with a representation of the step reference volume, an estimate of the volume of metered liquid received in the pipette tube, and the volume displaced by the metering piston face. [Figure 4B] FIG. 2 shows a graph of a conventional aspiration process with simple piston stroke control according to a target metered liquid volume to be metered. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0155] 1 to 3, a pipetting device according to the invention is shown generally at 10. The pipetting device comprises a pipette tube 12 formed by a cylinder 13, which is a tube portion fixed to the device, and a pipette tip 26 releasably connectable to the cylinder 13. The pipette tube 12 extends along a tube trajectory K which is formed as a linear tube axis. A piston 14 is received in the pipette tube 12 so that it can move along the tube trajectory K.

[0156] The piston 14 comprises two end caps 16, referenced only in FIG. 1, between which a number of permanent magnets 18 (three permanent magnets 18 in the present example) are received. The permanent magnets 18 are polarized along the tube axis K and arranged in pairs with like poles facing each other in order to obtain a magnetic field with high separation along the tube trajectory K. This arrangement results in a magnetic field starting from the piston 14, which is approximately homogeneous around the tube axis K, i.e. approximately rotationally symmetric with respect to the tube axis K, and which has a high gradient of field strength along the tube axis K, so that non-homogeneous polarization areas alternate with a high degree of separation along the tube trajectory K. Thus, a high position resolution is obtained when detecting the position of the piston 14 along the tube axis K, for example by means of a Hall sensor 19, which is shown only symbolically, and a very efficient coupling of the external magnetic field to the piston 14.

[0157] The end cap 16 is preferably made of a low-friction, graphite-containing material, as known, for example, from commercially available pistons from Airpot Corporation, Norwalk, Connecticut, USA. In order to be able to completely eliminate the small friction caused by the material, the cylinder 13 is preferably constructed as a glass cylinder, so that the graphite-containing material slides with extremely low friction on the glass surface as the piston 14 moves along the tube axis K.

[0158] The piston 14 thus forms the rotor of a linear motor 20, the stator of which is formed by coils 22 (only four coils are shown by way of example in the drawings) surrounding the pipette tube 12.

[0159] It should be clearly pointed out that Figures 1 to 3 are merely rough schematic longitudinal cross-sections of a pipetting device 10 according to the invention and should not be understood as being drawn to scale. Furthermore, when there are a plurality of elements, an arbitrary number of elements is shown, for example three permanent magnets 18 and four coils 22. In practice, the number of permanent magnets 18 and the number of coils 22 may be more or less than those shown.

[0160] The linear motor 20, or more precisely its coil 22, is operated by a control device 24 connected to the coil 22 in a signal transmitting manner. The transmission of an electric current for powering the coil and the generation of a magnetic field by the coil are also considered as signals. The control device 24 receives a pressure detection signal representative of the pressure of the working gas 34 in the pipette tube 12 from a pressure sensor 38, a position detection signal representative of the position of the pipette piston 14 from a Hall sensor 19, and a temperature detection signal representative of the temperature of the working gas 34 in a second operating area AB2 of the pipette tube 12, which will be mentioned below, from a temperature sensor 23. In FIG. 1, the temperature sensor 23 is hidden by the pipette piston 14 and is only visible in FIGS. 2 and 3.

[0161] A pipette tip 26 is releasably attached in a manner known per se to the metering end 13a of the cylinder 13, which extends the pipette tube 12 beyond the cylinder 13. The connection of the pipette tip 26 with the metering longitudinal end 13a of the cylinder 13 is likewise only indicated roughly and diagrammatically.

[0162] The pipette tip 26 defines a pipetting space 28 inside the pipette tip, which is accessible exclusively through a pipette opening 30 at the longitudinal end 26a remote from the connection.

[0163] In the example of an aspiration process shown in Figures 1 to 3, a quantity 31 of metering liquid 32 is received in the pipetting space 28. Figure 1 shows an operating state of the pipetting device 10 at the start of the aspiration process, in which the pipette tube 12, with its pipette tip 26 and its pipette opening 30, is exactly in contact with the liquid level 32a of the metering liquid container 40, whereby the working gas 34 is trapped between the pipette opening 30 and the metering piston surface 14a. In the example, the metering piston surface 14a is formed by the end face of the end cap 16, which points axially with respect to the tube trajectory K towards the metering opening 30.

[0164] In the case of the "air displacement" pipetting method discussed herein, a working gas 34 is constantly present between the piston 14 and the metered liquid 32, and the working gas 34 is used as a medium for transmitting force between the piston 14 and the metered liquid 32.

[0165] In the operating state shown in FIG. 1, there is a working gas dead volume V between the metering piston surface 14a and the pipette opening 30 closed by the metering liquid 32. T In the initial state of the pipetting device 10, the metering piston surface 14a is preferably located at the bottom dead center for the subsequent aspiration process. At the bottom dead center, the metering piston surface 14a is in a first operating area AB1, which extends from the pipette opening 30 to the axial start of the linear motor 20. In the first operating area AB1, the pipette tube 12 is directly wetted by the ambient atmosphere on the radial outside, so that the temperature T of the working gas 34 trapped in the first operating area AB1 is AB1 is the ambient temperature T ∞The pipetting space 28 is allowed to come into contact with the ambient atmosphere until just before the pipette opening 30 is placed on the liquid level 32a of the metering liquid 32. Therefore, in the operating state of the pipetting device 10 of FIG. AB1 is the ambient pressure p ∞ 1 shows the pipetting device 10 in a reference state for the ensuing aspiration process. The parameter values ​​mentioned are therefore reference values.

[0166] Structurally, the first working area AB1 is followed in the axial direction by a second working area AB2, which extends in the axial direction over the length of the linear motor 20 or over the arrangement of the coils 22 of the linear motor. The coil 22, which can be energized to drive the pipette piston 14 along the tube trajectory K, is a heat source and when moving along its extension, i.e. along the second working area AB2, it increases the temperature T AB2 is the temperature T AB1 The increased temperature T AB2 is detected by the temperature sensor 23 shown in Figures 2 and 3 or is known because, during continuous operation of the pipetting device 10, a constant elevated equilibrium temperature T AB2 This is because:

[0167] The metered liquid 32 and the material dependent parameters required to aspirate and dispense the metered liquid are unknown.

[0168] 1, a defined amount of metering liquid 32 is to be aspirated into the pipette tube 12. For this, the pipette piston 14 is moved in the aspirating direction, i.e. away from the pipette opening 30, along the tube trajectory K with a step frequency between 100 Hz and 50 000 Hz. The movement is controlled by the control device 24.

[0169] Through manual input, network-assisted data inquiry, or further sensors not specifically shown, the control device 24 queries the ambient pressure and the ambient temperature. Furthermore, the control device 24 reads out the number of pipette piston movement steps, which is stored in the data storage device of the control device 24 and which preferably depends on the target metered liquid volume to be aspirated. Alternatively, this number of pipette piston movement steps can be calculated by the control device 24 using a formula or can be entered into the control device 24 by manual input.

[0170] Given a known target metered liquid volume to be aspirated, the control device obtains, through querying a correspondingly filled data storage device or through calculation using a formula for each pipette piston movement step, a step reference volume as the incremental step reference volume to be advanced starting from the piston position at the beginning of the pipette piston movement step, knowing the size of the metering side piston face 14a, or as the cumulative step reference volume representing the final position at the end of the pipette piston movement step, knowing the size of the metering side piston face 14a. As a correction value, a step correction movement volume is initialized, for example with a value of 0.

[0171] The metering piston surface 14a displaces the target step movement volume away from the pipette opening 30 by means of the corresponding energization of the coil 22 in the first pipette piston movement step, corresponding to the sum of the step reference volume for this movement step and the step correction movement volume for this movement step. The step correction movement volume for this first step has, by way of example, an initialization value of 0, so that in this case the target step movement volume is the step reference volume.

[0172] Since the pipette piston movement step, beginning from the starting position of the metering side piston surface 14a, is located entirely within the first operating region AB1 of the pipette tube 12, the state change of the trapped working gas 34 brought about by the movement of the metering side piston surface 14a is treated like an isothermal state change.

[0173] The pressure sensor 38 detects the pressure p of the trapped working gas 34. AB1 is detected, and the pressure is reduced by the initial ambient pressure p ∞ Less than.

[0174] Using the above equation 10', or the above equation 11 if leakage losses are to be considered, we can calculate the known V Kolben , in this case V, which corresponds to the first step reference volume Kolben and since the piston face 14a moves exclusively in the first operating region AB2 V Kolben = 0 and the measured pressure p of the trapped working gas 34 AB1 is used to calculate the amount V of metered liquid present in the pipette tube 12 after the first pipette piston movement step. liquid is evaluated as the estimated volume.

[0175] Step reference volume and estimated volume V liquid , a difference value is formed, for example by difference formation, which is calculated by dividing the step reference volume by the estimated value V liquid It represents the difference between the values.

[0176] Based on the difference value, a step correction movement volume is calculated as a correction value through a PID control known in principle, with which the step reference volume of the subsequent pipette piston movement step is corrected relative to the target step movement volume. For this purpose, the difference value is converted into the step correction movement volume by means of a proportional conversion element, a differential conversion element and an integral conversion element. Each conversion element may be weighted by a weighting factor determined by experiment.

[0177] The counter of the pipette piston movement steps is incremented by one and, if the maximum number of pipette piston movement steps is still not reached, the step reference volume assigned to the next pipette piston movement step is used and corrected with the predetermined step correction movement volume against the target step movement volume, for example by forming a sum or difference. The metering side piston surface 14a is then moved in accordance with the target step movement volume, taking into account the size of its surface. That is to say, the target step movement volume is divided by the value of the size of the metering side piston surface 14a, and the displacement path of the piston surface 14a along the tube trajectory K is obtained.

[0178] As mentioned above, the pressure p of the trapped working gas 34 AB1 is newly found and can be calculated from equation 10', or from equation 11 if leakage losses are to be considered, by the known V Kolben and AB2 V Kolben = 0 and the measured pressure p of the trapped working gas 34 AB1 is used to calculate the amount V of metered liquid present in the pipette tube 12 after the pipette piston movement step has been performed. liquid is estimated.

[0179] The above step reference volume and the estimated value V liquid A new difference value is formed from this difference value, from which the above-mentioned further step correction movement volume is calculated, which is again used to determine the next target step movement volume, followed by an increment of the step counter and a new movement of the metering piston surface 14a.

[0180] This sequence is repeated in the described manner until the metering side piston surface 14a reaches the boundary between the first operating area AB1 and the second operating area AB2, so that further movement of the piston surface 14a no longer takes place in the first operating area AB1, but in the second operating area AB2.

[0181] Next, when the metering side piston surface 14a is moved in the second operating area AB2, the movement of the piston surface 14a causes a displacement of the trapped working gas 34 between the first operating area AB1 and the second operating area AB2, so that the above-mentioned progression is basically maintained, but the estimated value V liquid In the above-mentioned equation 10, equation 10', equation 10" or equation 11, which may be used to determine AB2 V Kolben This allows the displacement of the working gas between the first and second operating areas AB1 and AB2 and the temperature change thereof to be taken into account in addition to the pressure change.

[0182] That is, the metering side piston surface 14a is displaced again according to the last calculated target step movement volume. AB1 is detected by the pressure sensor 38, and the temperature T AB2 is detected by the temperature sensor 23. The temperature T AB1 is assumed to be approximately constant, and from the above considerations, the ambient temperature T ∞ is considered to be the same as

[0183] The detected parameters are then used to calculate an estimate V for the volume of metered liquid 32 received in the pipette tube 12 based on Equation 10, Equation 10', or Equation 10'', or Equation 11. liquid is determined. By comparing the estimated value with the step reference volume assigned to the latest pipette piston movement step, a difference value is determined, and based on the difference value, a step correction movement volume is determined using a PID control, the principles of which are known as described above. Using this step correction movement volume, the step reference volume is corrected relative to the target step movement volume.

[0184] If after the increment of the step counter the maximum number of steps for the pipetting process has still not been reached, the next pipette piston movement step is performed with the target step movement volume as the target reference value for the movement of the metering side piston face 14a.

[0185] This progression is repeated until a certain maximum number of steps for the pipetting process is reached or until the difference value, possibly a predetermined number of consecutive steps, falls below a predetermined threshold so that the estimated volume of metered liquid 32 present in the pipette tube, determined by Equation 10, Equation 10' or Equation 10" or Equation 11, matches the target metered liquid volume with sufficient accuracy.

[0186] Advantageously, the progression of the step reference volume through the number of pipette piston movement steps provided for the pipetting process is selected to be decreasing rather than linear, i.e. the increase in the value of the cumulative step reference volume or the cumulative incremental step reference volume, depending on which, is greater at the beginning of the pipetting process than towards the end of the pipetting process. In this case, the value of the step reference volume for at least the last 30% of the pipette piston movement steps of the pipetting process does not change by more than 10% with respect to the greater of the two comparison values. The advantage of the decreasing progression of the step reference volume is that towards the end of the pipetting process, the influence of the correction value determined from the difference value on the movement of the metering side piston surface 14a becomes dominant. Thus, towards the end of the pipetting process, for example during the last 30% of the pipette piston movement steps, the influence of different flow properties of different metering liquids can be eliminated by the determination of the difference value and the correction value (step correction movement volume) obtained from the difference value.

[0187] Different metered liquids will converge to the desired target metered liquid volume at different speeds depending on the influencing variables related to pipetting such as viscosity, density, surface tension, wettability, behavior of the pipette tube towards the material, etc. By choosing the number of pipette piston movement steps large enough, it can be ensured that metered liquids with relatively high density and high viscosity can also be pipetted accurately without knowing their exact liquid parameters.

[0188] Figure 4A shows a graph of an aspiration process as an example of a pipetting process according to the present invention. The x-coordinate of the coordinate system in Figure 4A represents time in seconds, i.e., 0 to 16 seconds, and the y-coordinate represents volume in microliters (μl), i.e., -5 μl to 20 μl.

[0189] The target metered liquid volume to be aspirated is 10 μl. The metered liquid to be aspirated is glycerin.

[0190] With reference number 42 the progression of the step reference volume is shown as an accumulated value. Up to t=5 seconds after the start of the aspiration process it has a value of 0 μl and in the time region from 5 to 10 seconds it increases linearly from 0 μl to the target metered liquid volume of 10 μl. Thus the pipetting process only starts at t=5 s. In the time region from 10 to 15 seconds the step reference volume continues to have the value of the target metered liquid volume of 10 μl. The time region from 5 to 10 seconds thus forms a first step region 44 in the above sense, in which the accumulated step reference volume increases to at least 95% of the target metered liquid volume. The region from 10 to 15 seconds forms a second step region 46 in the above sense, in which the accumulated step reference volume does not leave the region from 95% to 105% of the target metered liquid volume. More precisely, the cumulative step reference volume increases from 0% in the first step region 44 to exactly 100% of the target metered liquid volume and remains at exactly 100% of the target metered liquid volume in the second step region 46 .

[0191] In FIG. 4A, reference numeral 48 denotes the volume displaced by metering end 14a during the pipetting process.

[0192] Starting from the zero position at the beginning of the pipetting process, the volume displaced by the metering end face 14a is initially negative, i.e., the metering end face 14a approaches the pipette opening 30 in the dispensing direction in order to resist the inflow of glycerol into the pipette tip 26 caused by capillary forces.

[0193] In fact, the volume difference 50 between the volume displaced by the metering side end face 14a, which corresponds to the movement volume of the metering side end face 14a and the cumulative target step movement volume, and the step reference volume is the step corrected movement volume calculated as described above.

[0194] Reference numeral 52 denotes an estimate of the metered liquid volume received in the pipette tube 12 or in the receiving space 28, calculated as described above.

[0195] As shown in FIG. 4A, first the movement of the pipette piston 14 simply prevents the capillary flow of glycerin into the pipette tip 26 according to a criterion set by the step reference volume 42 .

[0196] Next, if the value of step reference volume 42 begins to increase at the 5 second point, the metered liquid will remain below step reference volume 42 for the time being, but if step reference volume 42 remains consistently at the reached target metered liquid volume at the 10 second point, it will exceed the step reference volume.

[0197] The second step area 46 following the first step area 44 is used, as already described in detail, to correct the tendency of the metered liquid to overflow or slip by a corrective movement of the metering end face 14a after the target metered liquid volume has been received in the receiving space 28. Since the target metered liquid volume has already been received in the receiving space 28 to a large extent in the first step area 44, the volume of each target step movement of the metering end face 14a is smaller in the second step area 46 than in the first step area 44, which generally results in a high metering accuracy of the pipetting process. In the illustrated example, the second step area 46 continues with approximately the same length as the first step area 44 and therefore includes approximately the same number of movement steps of the pipette piston 14.

[0198] FIG. 4B shows how glycerin behaves during a purely path- and time-controlled suction movement of the pipette piston 14 when the pipette piston 14 is raised to the desired target metered liquid volume, taking into account the surface area of ​​the metering end face 14a.

[0199] In this figure, the x-coordinate again represents time in seconds and the y-coordinate represents volume in μl.

[0200] In Fig. 4B, the target motion trajectory of the metering end surface 14a is shown with reference numeral 42'. The target motion trajectory exactly coincides with the cumulative step reference volume in Fig. 4A.

[0201] Reference numeral 48' denotes a motion curve of the metering end face 14a which follows the target motion trajectory 42' in a path- and time-controlled manner. It is technically no problem to control the metering end face 14a in a path- and time-controlled manner according to the target motion trajectory 42', so that the metering end face 14a follows the target reference value very accurately.

[0202] With reference numeral 52' the metered liquid volume received by the pipette tip 26 is shown over time. It can be very clearly seen from the graph of Fig. 4B that, without any corresponding trimming by the pipette piston 14, at approximately t=1 s the glycerin starts to flow through the pipette opening 30 into the receiving space 28 of the pipette tip 26, driven only by capillary forces. At t=1 s the pipette opening 30 is immersed in the metered liquid 32 in both cases, i.e. Fig. 4A and Fig. 4B.

[0203] At time t=5s, with the start of the piston movement, more glycerin also begins to flow into the receiving space 28 of the pipette tip 26, but this time driven by the negative pressure created in the working gas 34 by the pipette movement relative to the ambient pressure.

[0204] After the piston movement ends at time t=10 s, glycerin continues to flow into the receiving space 28 through the pipette opening 30 until the negative pressure in the pipette tube 12 (and thus in the receiving space 28) decreases to a degree that is approximately in force equilibrium with the liquid column 29 formed by the glycerin in the receiving space 28. However, this results in only about less than 8 μl of glycerin being received into the receiving space 28, even though the pipette piston 14, as the driving mechanism for receiving glycerin, performs a movement of 10 μl.

[0205] In a conventional pipetting system, a liquid class would be stored in the data storage device of the control device 24. Glycerin would be assigned to that liquid class, and a coefficient would be derived from that liquid class by which the target moving volume of the pipette piston 14 must increase starting from the desired 10 μl, so that the pipette tip 26 will have received the desired 10 μl of glycerin at the end of the piston movement. The coefficient must be determined empirically in the laboratory.

[0206] As a comparison of Figures 4A and 4B shows, the present invention makes it possible to pipette a desired target metered liquid volume with high precision based on the described volume-based control without knowing the liquid class of the metered liquid and without knowing the specific flow characteristics of the metered liquid, and even to pipette with high precision in a shorter time than pipette piston 14 with conventional path or path-and-time controlled motion. [Explanation of symbols]

[0207] 10 Pipetting device 12 Pipette Tubes 13 Cylinder, tube part fixed to device 13a Measuring side end 14 Pipette piston 14a Metering side piston surface 16 End Cap 18 Permanent Magnets 19 Hall sensors, position detection devices 20 Linear motor, drive unit 22 Coil 23 Temperature Sensor 24 Control device 26 Pipette Tips 26a Longitudinal end 28 Pipetting space, receiving space 29 Quantity of liquid measured in the receiving space, liquid column 30 Pipette openings 31 quantity 32 Metering liquid 32a Liquid level 34 Working Gas 38 Pressure sensors, pressure detection devices 40 Measuring liquid container 42 Step Reference Volume 42' Target motion trajectory 44 First Step Area 46 Second Step Area 48 Volume displaced by metering end face 14a during the pipetting process 48' Motion curve of the measuring side end surface 14a 50 Volume difference 52 Estimation of Measured Liquid Volume 52' Metered liquid volume received in pipette tip 26 AB1 First operating area AB2 Second operating region K Tube orbit, tube axis p AB1 Detected working gas pressure p ∞ Working gas reference pressure T AB2 operating temperature T ∞ Reference temperature AB1 V Kolben The portion of the volume displaced by the metering piston face that is located in the first operating region AB2 V Kolben The volume of the working gas corresponding to the volumetric part located in the second operating region of the piston movement V1: Volume of working gas trapped in the pipette tube V Kolben Displacement Volume V T Working gas volume V liquid An estimate of the amount of metered liquid present in the receiving space V sys,rest System specific residual volume

Claims

1. A pipetting device (10) for pipetting, i.e. for aspirating and / or dispensing, a metered liquid (32) with a working gas (34), the pipetting device (10) comprising: - a pipette tube (12) extending along a tube axis (K), a pipette piston (14) movable within said pipette tube (12) along said tube axis (K); a receiving space (28) for receiving a metering liquid (32), said receiving space (28) extending in said pipette tube (12) along said tube axis (K) from a pipette opening (30) at one end to a metering side piston face (14a) of said pipette piston (14) facing said pipette opening (30) at the other end, said working gas (34) being received in said pipette tube (12) in direct contact with said metering side piston face (14a), said working gas reference volume (VT) being determined by the volume of said working gas (34) present in said receiving space (28) under a working gas reference pressure (p∞); a drive (20) connected to said pipette piston (14) in a force-transmitting manner, said drive (20) being adapted to displace said pipette piston (14) along said tube axis (K); a position detection device (19) for detecting the position of the pipette piston (14) along the tube axis (K) and for outputting a position detection signal representative of the detected position; a pressure detection device (38) for detecting the pressure of the working gas (34) in the pipette tube (12) and for outputting a pressure detection signal representative of the detected pressure; and a control device (24), Contains The control device (24) controls the drive device (20) to said pressure detection signal, the reference pressure of the working gas (p∞), and - the determined target metered liquid volume to be pipetted, a control device (24) configured to perform the piston movement required for pipetting the target metered liquid volume in a plurality of successive movement steps, said control device (24) being further configured to operate taking into account a pressure change induced change in said reference volume of working gas (VT) brought about by a piston movement according to the pipette tube (12) has a first operating area (AB1) and a second operating area (AB2), the first operating area (AB1) starting at the pipette opening (30) and extending from the pipette opening (30) into the tube of the pipette tube (12), the first operating area (AB1) being located in an ambient atmosphere having a substantially constant ambient temperature, the temperature of the first operating area (AB1) being a reference temperature, the first operating area (AB1) being located in a reference temperature area with a lower known reference temperature, the second operating area (AB2) being along a heat source of the pipette device (10), the temperature of the second operating area (AB2) being an operating temperature, the second operating area (AB2) being located in an operating temperature area with a higher known operating temperature than the reference temperature area, and the control device (24) after a first pipette piston movement step and for a subsequent pipette piston movement step: determining a first correction variable representative of the pressure change-induced volume change of the portion of the working gas volume trapped in the pipette tube (12) located in the first operating area (AB1) on the basis of the position detection signal, the pressure detection signal and the reference pressure of the working gas (p∞); determining a second correction variable representative of the pressure and temperature change induced volume change of the portion of the working gas volume trapped in the pipette tube (12) located in the second operating area (AB2) based on the position detection signal, the pressure detection signal, the working gas reference pressure (p∞), the known operating temperature and the known reference temperature; The target step movement volume of the pipette piston (14) is - the step reference volume to be assigned to the subsequent pipette piston movement step, - the displacement volume (VKolben) of the metering piston surface (14a) of the pipette piston (14) so ​​far during the ongoing pipetting process, said first correction variable, and said second correction variable, and actuating the driver in accordance with the determined target step movement volume.

2. 2. The pipetting device according to claim 1, wherein the control device is configured to determine, for a subsequent pipette piston movement step, an estimate for the amount of metering liquid present in the receiving space on the basis of a displacement volume so far of the metering side piston surface of the pipette piston during an ongoing pipetting process, the first correction variable and the second correction variable, compare the determined estimate with the step reference volume and determine the target step movement volume on the basis of the comparison result.

3. 3. The pipetting device (10) of claim 2, characterized in that the control device (24) is configured to perform successive pipette piston movement steps until a difference value between the particular estimated value and the step reference volume falls below a predetermined difference threshold.

4. 3. The pipetting device (10) of claim 2, wherein the control device (24) is configured to perform a predetermined number of the pipette piston movement steps.

5. 5. The pipetting device according to claim 2, wherein the control device is configured to calculate a difference value between a particular estimated value and the step reference volume and to determine a part of the step corrected moving volume that is proportional to the difference value, or / and an integral part of the step corrected moving volume taking into account the sum of the difference value and at least one preceding difference value, or / and a differential part of the step corrected moving volume taking into account the difference between the difference value and a preceding difference value.

6. 2. The pipetting device (10) of claim 1, wherein the control device (24) is configured to determine the target step movement volume based on the step reference volume and the step correction movement volume.

7. 7. The pipetting device (10) according to claim 6, characterized in that the control device (24) is configured to determine the target step movement volume as a step reference volume corrected by the step correction movement volume.

8. 8. The pipetting device (10) of claim 1, wherein the control device (24) is configured to perform 100 or more pipette piston movement steps per second, and wherein the control device is configured to perform less than 100,000 pipette piston movement steps per second.

9. 9. The pipetting device (10) of claim 8, wherein the control device (24) is configured to perform 1000 or more pipette piston movement steps per second.

10. 10. The pipetting device (10) of claim 9, wherein the control device (24) is configured to perform 10,000 or more pipette piston movement steps per second.

11. 11. The pipette device (10) according to claim 1, wherein the control device (24) is configured to read out the step reference volume assigned to a subsequent pipette piston movement step from a data storage device in accordance with the target metered liquid volume and / or to calculate the step reference volume based on the target metered liquid volume.

12. the step reference volume is an incremental step reference volume, the cumulative value of the step reference volume through an increasing number of steps having a first step region (44) in which the cumulative value increases from an initial value to at least 95% of the target metered liquid volume, and a second step region (46) following the first step region (44) in which the cumulative value does not leave the range of 95% to 105% of the target metered liquid volume; or the step reference volume is an absolute step reference volume, the value of the absolute step reference volume through the increasing number of steps having a first step region (44) in which the value of the absolute step reference volume increases from an initial value to a value of at least 95% of the target metered liquid volume, and a second step region (46) following the first step region (44) in which the value of the absolute step reference volume does not leave a range of 95% to 105% of the target metered liquid volume, 12. A pipetting device (10) according to any one of claims 1 to 11, characterized in that the second step region (46) has at least the same number of steps as the first step region and / or lasts for at least the same length of time as the first step region.

13. The control device (24) calculates the determined target step motion volume based on the following three parameters: - the target final position of the metering side piston surface (14a) at the end of a subsequent pipette piston movement step; - the target displacement speed of the metering piston surface (14a) during the subsequent pipette piston movement step, and - the time length of the subsequent pipette piston movement step, 13. A pipetting device (10) according to any one of claims 1 to 12, characterized in that it is arranged for the determination to be made by quantification of at least two of the following parameters:

14. 14. The pipette device (10) of claim 1, wherein the control device (24) is configured to form the second correction variable based on the portion of the working gas (34) located in the second operating region (AB2) and a product consisting of a pressure quotient and a temperature quotient, the pressure quotient being the quotient consisting of the detected working gas pressure (pAB1) and the working gas reference pressure (p∞), and the temperature quotient being the quotient consisting of the operating temperature (TAB2) and a reference temperature (T∞).

15. 15. The pipetting device (10) according to claim 1, characterized in that the drive (20) comprises a linear motor, the rotor of which is the pipette piston (14), the drive (20) comprises a number of energizable coils (22), which are arranged radially outside the pipette tube (12) along a drive section, and the second operating area (AB2) comprises or is the drive section.

16. 16. The pipetting device (10) according to claim 1, wherein the first operating area (AB1) extends starting from the pipette opening (30) in a direction towards the pipette piston (14).

17. 17. The pipetting device (10) according to claim 16, characterized in that the first operating area (AB1) extends from the pipette opening (30) in a direction towards the pipette piston (14) to the second operating area (AB2).

18. 18. The pipetting device (10) according to claim 1, characterized in that the pipetting device (10) has a temperature sensor (23) for detecting an operating temperature (TAB2), the temperature sensor outputting an operating temperature signal representative of the operating temperature (TAB2).

19. 19. The pipette device (10) of claim 1, wherein the pipette tube (12) comprises a tube portion (13) fixed to the device with a connecting structure and a pipette tip (26) releasably connected to the connecting structure, the pipette tip (26) having the pipette opening (30).

20. 20. The pipetting device (10) according to any one of claims 1 to 19, characterized in that the control device (24) is configured to determine a third correction variable representative of leakage of the pipette tube (12), and the control device (24) is configured to determine the target step movement volume of the pipette piston (14) additionally based on the third correction variable.

21. 21. The pipetting device (10) of claim 20, wherein the third correction variable represents a leakage volume, and the control device (24) is configured to determine the third correction variable based on the pressure detection signal and a time length.

22. 22. The pipette device (10) of claim 21, characterized in that the third correction variable represents a leakage volume, and the control device (24) is configured to determine the third correction variable based on the pressure detection signal and the length of the pipette piston movement step.

23. A pipette device (10) according to any one of claims 20 to 22, characterized in that the control device (24) is configured to determine the third correction variable also based on a leakage parameter, and the control device (24) is further configured to determine the leakage parameter by performing detection of the time-dependent change in the initially determined working gas pressure, which is influenced by leakage, based on a manual control input or by a predetermined automatic control.

24. A method for accurately pipetting a metered liquid (32) using a pipetting device (10), independent of the flow properties or / and wetting properties of said metered liquid (32), wherein a pipette tube (12) has a first operating area (AB1) and a second operating area (AB2), said first operating area (AB1) starting at a pipette opening (30) and extending from said pipette opening (30) to an interior of said pipette tube (12), said first operating area (AB1) being located in an ambient atmosphere having a substantially constant ambient temperature, said temperature of said first operating area (AB1) being a reference temperature, and in said first operating area (AB1) a known temperature is maintained. a reference temperature (T∞) of said pipetting device (10) being located in a lower reference temperature region, said second operating region (AB2) being along a heat source of said pipetting device (10), said temperature of said second operating region (AB2) being an operating temperature, said second operating region (AB2) being located in an operating temperature region higher than said reference temperature region, said method carrying out a stepwise displacement of a pipette piston (14) movably received in said pipette tube (12), said method comprising the steps of: - detecting the pressure (pAB1) of the working gas (34), - detecting the position of the pipette piston, determining a first correction variable representative of a pressure change-induced volume change of a first portion of a working gas volume (V1) contained in the pipette tube (12) located in the first operating area (AB1) on the basis of the detected position of the pipette piston, the detected pressure (pAB1) of the working gas (34) and a working gas reference pressure (p∞); determining a second correction variable representative of the pressure- and temperature-induced volume change of the portion of the working gas volume (V1) contained in the pipette tube (12) located in the second operating area (AB2), based on the detected position of the pipette piston, the detected pressure (pAB1) of the working gas (34), the working gas reference pressure (p∞), the known operating temperature (TAB2) and the known reference temperature (T∞); - determining an estimate (Vliquid) for the metering liquid (32) present in the receiving space (28) on the basis of the detected position of the pipette piston, the previous position of the pipette piston, the first correction variable and the second correction variable, - determining or retrieving from a data storage device the step reference volume assigned to the subsequent pipette piston movement step; - comparing said estimated value (Vliquid) with said step reference volume; - determining a target step movement volume for the displacement of the pipette piston in a subsequent pipette piston movement step; and - displacing the pipette piston (14) by the target step movement volume, A method comprising:

25. Method according to claim 24, characterised in that the pipetting device (10) is a pipetting device (10) according to any one of claims 1 to 23.

26. Steps below: - detecting the operating temperature (TAB2) in the second operating area (AB2) of the pipette tube (12), 26. The method of claim 24 or 25, further comprising:

Images