EP0562358B2 - Process for the correction of the volume error at the design of a pipetting system - Google Patents

Description

The invention relates to a method for correcting the volume error in the design of a pipetting system with a piston stroke and a pipette tip that can be plugged in.

From DE-PS 25 26 296 a pipetting system of the aforementioned type is known, the Kolbenhubpipette has a cone for attaching the pipette tip. The pipette tip has one starting from the tip opening strongly conical and then up to the slip on a weaker conical section based on the inner contour on. Such a pipette tip when immersed in a deep and especially slim vessel has the disadvantage that it carrying a pipette wall contact with the vessel get and be contaminated. This is basically a pipette tip not the case, according to EP-A-0 182 943 from a tip opening a long cylindrical nose, then a strong conical and then a Has slip-on.

From the brochure "Comforpette 4700, Varipette 4710 and Comfortip cristal the new Pipetting system for 0.5 to 10 μl ", edition 1985 of Eppendorf Gerätebau Netheler & Hinz GmbH is a pipetting system consisting of the pipette Varipette® 4710 and the Pipette tips Comfortips® cristal known, in which the piston of the pipette in the Protrudes pipette tip and thereby reduces the air cushion to a minimum. Turning the control knob on the pipette determines the volume Work shift changed. The working stroke causes the recording and delivery of the Pipetting. The pipette tips Comfortips® cristal are adjacent to one Shaft into which protrudes the piston, a shaped into a wider cone tip.

In the pipetting systems mentioned, the adjustment range extends more absorbably Amounts of liquid each from the tip opening over all different sections away from the pipette tip. If a small piston stroke is set, fluid is only in the strongly conical (or cylindrical) initial area sucked in. For larger Piston stroke becomes the weaker conical (or the conical and then the cylindrical) Area reached. The pipetting systems have in common that they interact with an adjustable piston stroke pipette to accommodate fluid volumes that are unacceptable may differ from the indicated liquid volume, the deviation over the Setting range is possible in different ways. Therefore, the slope of a Spindle of the piston stroke pipette for the adjustment of the piston stroke on a trial basis determines that the correctness deviation between recorded and displayed liquid volume can deviate, with the deviation is possible differently over the adjustment range. Therefore, the pitch of a spindle of Kolbenhubpipette for the adjustment of the piston stroke tentatively determined so that the corrective deviation between recorded and displayed volume of liquid over the entire adjustment range is bearable. The resulting accuracy deviations are accepted and are possibly due to subsequent measurements ascertainable.

This is beyond the neglect of corrective deviations of unknown size over the setting range disadvantageous that the vote of Kolbenhubpipette and pipette tip changing the pipette tip difficult. Should e.g. a Kolbenhubpipette with a pipette tip according to DE-PS 25 26 296 with a pipette tip be equipped according to EP-A-0 182 943, so with the given spindle pitch is no longer guaranteed that the Correctness deviations over the setting range are bearable.

U.S. Patent No. 5,024,109 already discloses a method and apparatus for correcting the failure of a piston-stroke pipette. The height of the liquid in the pipette tip for the desired liquid volume, the Changes in the dead volume due to the liquid column in the tip and the required piston displacement determined for the desired liquid volume and the change in the dead volume. The height of the liquid column for a desired volume is determined by the tip geometry, especially for a conical tip. The change The dead volume is determined on the basis of hydrostatic considerations related to the height of the liquid column tie. The piston displacement becomes the sum of the desired volume and the change in the dead volume determined. This technique uses an electromechanical pipette connected to a control computer is. Before use, data of the pipette tip, the pipetting system and the liquid must be entered into the computer be entered. This pipetting technique is expensive.

On this basis, the invention has the object, the pipetting system of the type mentioned above improve so that over the entire adjustment range of pipetting liquid to be taken over easily and accurately can be worked. Furthermore, the pipetting system is intended to replace the piston stroke pipettes or pipette tips while continuing to work accurately and simply.

The solution to this problem is specified in claim 1. Advantageous embodiments are in the subclaims to find.

The invention is based on the finding that pipette correction factor, cross-sectional profile along the tip, Dead volume and, if necessary, zero offset of the display for the amount of the corrective deviations the setting range are relevant. By matching these influencing factors to each other, it is therefore possible in the Adjustment range for each liquid volume taken a given accuracy deviation from the displayed To realize liquid volume, which can also be zero. In contrast to conventional pipetting systems are no longer corrective deviations by selecting the spindle pitch according to the invention according to specification minimized the remaining predictors, but all o.g. Influencing variables coordinated so that predetermined Correctness deviations are achieved. In such a pipetting system is then for each indicated liquid volume the exact value of the liquid volume taken known. Compared to the previously known electronic pipette with control computer, a calculation of the piston stroke is unnecessary for each pipetting process, to achieve the given accuracy deviation. At best, an adaptation to the respective liquid density to be required. This can be done by exchanging tips, changes in the dead volume or the pipette correction factor take place in accordance with the respective liquid. If the correctness deviations from zero can deviate and be pipetted without error, the piston stroke can always be adjusted by means of the display that the volume of liquid absorbed corresponds to the intended volume. Furthermore, it is possible according to the invention Adjust pipette tips on conventional piston pipettes so that given corrective deviations be achieved. In this way, if necessary, a pipetting system with an extended pipette tip equipped, which avoids wall touching and concomitant contamination. It's the other way round too possible, predetermined accuracy deviations by tuning a piston stroke pipette on existing pipette tips sure.

The correctness deviation can be defined in various ways. For practical handling the specification of a constant absolute or relative accuracy deviation is particularly favorable, because then the Difference of the recorded liquid volume is the easiest to determine.

If a conventional pipetting system in particular by replacement of a pipette tip in an inventive can be converted, a vote for each recorded volume of liquid a predetermined accuracy deviation from absorbed liquid volume of the conventional pipetting system instead of indicated volume of fluid conditionally. Then the differences are pipetted Volume of liquid from the conventionally pipetted volume at the same Ad known. This knowledge may be changed for comparative studies Be interesting pipette tip.

The liquid level always rises in the adjustment range of absorbable liquid volumes up into a cylindrical working area of the pipette tip. To compensate for Cross - sectional changes including discontinuities in the initial area of the Pipette tip, the vote can be a zero offset of the displayed Include fluid volume with respect to the piston stroke.

In practice, sometimes values of pipette correction factor, dead volume and where appropriate zero offset specified, especially if the Pipetting system by adapting a pipette tip to an existing one Kolbenhubpipette to be realized. To achieve the given Corrective deviation can then be a vote of the cross-sectional profile along the Peak to meet the predetermined factors.

The working area of the pipette tip is substantially cylindrical. Then, in particular, a constant Absolute accuracy deviation over the entire range of absorbable liquids realized away become. For a small tip opening and a sufficiently large volume range for recording adjustable Liquid quantities may have a gradually widening transition region. The transition area preferably has a transition radius or transition cone, wherein the cone angle to avoid unwanted Effects (fountain effect) is kept as small as possible. The transition region is preferably one Subordinate workspace. Especially for use with relatively long and slim vessels has the pipetting system a pipette tip with a thin aspiration tube adjacent to the tip opening. Then the Tip also prefers an expanding transitional as well as a downstream workspace.

To match the pipetting system to different liquid densities or pressures, pipette correction factor may be be adjustable. This is in the simplest case by replacing a spindle for adjusting the Piston strokes feasible. Also comes for a display device with a spreadable scale into consideration.

For the above purpose, a zero offset of the display can also be realized by means of similar measures become.

Furthermore, an adjustable dead volume can be provided. This is especially the replacement of pipette tips possible, with a pipette tip designed for lighter liquids with the same diameter longer must become. To change the dead volume can also intermediate pieces between Kolbenhubpipette and pipette tip be usable.

According to the invention, a vote of the influencing variables to achieve the predetermined corrective deviations done experimentally. It can first for each recorded fluid volume a corrective deviation be specified by the indicated liquid volume. These deviations are now through Adjustment of pipette correction factor, cross-sectional profile along the tip and dead volume to realize each other. Since the pipette correction factor and the dead volume are usually no longer changed after making the selection, First, the values for these influence parameters are at least provisionally determined, with an orientation to Values of known pipetting systems is possible. Then remains as the last predictor of cross-sectional shape along the pipette tip, with the specified accuracy deviations can be ensured. For that result For example, by experiments unique values, if the other influencing factors have been previously chosen. If the cross-sectional profile found could not be realized, the predetermined influencing variables would have to be corrected or even specify another correctness deviation.

Because of the expense of an empirical tuning, the following physical-mathematical model is preferably used for arithmetical tuning: The piston stroke volume V _{piston of} a piston stroke pipette is larger than the volume of liquid V liquid absorbed by the volume _{error ΔV.} , V piston = V Liq. + ΔV

The volume error results from the fact that the liquid column on the air cushion between liquid level and Piston (= "dead volume") hangs and pulls down, whereby the air cushion is increased accordingly.

V_t =

Luftpolster, Totvolumen von Pipette und Spitze

P_o =

Außendruck, atmosphärischer Druck

Δp =

Druckänderung entsprechend hydrostat. Druck der Flüssigkeitssäule in der Pipettenspitze

aus (B) folgt: po po - Δp Vt = Vt + ΔV und:

weiter - ΔV = ΔPPo - ΔP Vt und unter Berücksichtigung, daß Δp << p_o
ergibt sich: ΔV = Δppo Vt * The changes in volume and pressure in the air cushion can be described from the dead volume of the pipette and the tip at ambient pressure using a thermal equation of state. For the calculation of ΔV it is assumed: ideal gas, T = const.
thus: p · V = const.
or: p O · V t = (p O - Δp) · (V t + ΔV) With:

V _t =

Air cushion, dead volume of pipette and tip

P _o =

External pressure, atmospheric pressure

Δp =

Pressure change according to hydrostat. Pressure of the liquid column in the pipette tip

from (B) follows: p O p O - Δp V t = V t + ΔV and:

further - ΔV = .DELTA.P P O - ΔP V t and taking into account that Δp << p _o
surrendered: ΔV = Ap p O V t *

ρ =

spezifisches Gewicht

g =

Erdbeschleunigung

Dead volume and atmospheric pressure are assumed to be known. The pressure change Δp corresponds to the hydrostatic pressure of the liquid column of height h: p = ρ. g. H With:

ρ =

specific weight

g =

acceleration of gravity

mit
Q (y) = Querschnitt im Abstand y von SpitzenöffnungThe height h of the liquid column is determined by the pipetted liquid volume and the cross-sectional profile of the pipette tip:

With
Q (y) = cross section at the distance y from the tip opening

From (A), (C) and (D) follows: V piston = V Liq. + ρ gh p O V t

With (E) and (F) can be the dependence of the piston stroke V _piston from the recorded liquid volume V _liquid. calculate, if necessary, with the help of numerical methods.

a =

Pipettenkorrekturfaktor

b =

Nullpunktsverschiebung

The piston stroke is limited by stops whose distance can be adjusted by means of a spindle which is coupled to the display device. Consequently, between V _piston and the indicated liquid volume V _Anz. a linear relationship: V piston = a · V Num. + b With

a =

Pipette correction factor

b =

Zero Offset

For the relationship between a and spindle pitch S: S (mm / revolution) = Counter calibration (μl / revolution) Piston cross-section (mm 2 ) · A

From (F) and (G) follows: V Num. = (V Liq. + ρ gh p O · V t - b) · 1 a

This results in the absolute accuracy deviation R _abs. : R Section. = V Liq. - (v Liq. + ρ gh p O · V t - b) · 1 a With: R Section. = V Liq. - V Num.

For the relative accuracy deviation R _rel. applies: R rel. = R Section. / V Liq. With: R rel. = V Liq. - V Number V Liq. ,

Q =

const.

With (I) or (J) and (E) now pipette _{correction factor} a, zero offset b, cross-sectional shape Q (y) and dead volume V _t are coordinated so that each liquid volume recorded V _liquid. a predetermined accuracy deviation R _abs. or R _rel. assigned. Appropriately, one will specify the corrective deviations from the indicated liquid volume, at least provisionally select pipette correction factor, zero offset and dead volume and then calculate the cross-sectional profile. On the other hand, the specification of special cross-sectional profiles along the tip is possible. Specifically, with a cylindrical pipette tip, from (E) follows: V Liq. = Q · h With:

Q =

const.

From (I) and (K): R Section. = V Liq. - (v Liq. + ρ g V Liq. p O · V t Q - b) · 1 a

If the course of the accuracy deviation is specified, the relationship between the dead volume, the pipette correction factor and the zero offset follows from this. If the absolute accuracy deviation R _abs. for all V _liquid. be constant, must apply: O = 1 - 1 a - ρg p O · V t Q · a and R _abs. = b / a

With the formula set (L) all determinants can be defined. There are only sizes for two specify the influence factors of dead volume, pipette tip cross section, pipette correction factor and zero offset.

Fig. 1

Zusammenwirken von Pipettenspitze und Kolbenhubpipette in einem Diagramm mit der Differenz von (angezeigtem) Flüssigkeitsvolumen und Kolbenhub auf der Ordinate und dem Kolbenhub auf der Abszisse;

Fig. 2

Pipettenspitzen P₁, P₂ und P₃ zu den Kurven A, B und C in Fig. 1;

Fig. 3

eine Pipettenspitze in stark vergrößertem, vierfach abgebrochenem Längsschnitt;

Fig. 4

einen oberen Abschnitt einer kolbenbetriebenen Pipette; und

Fig. 5

einen unteren Abschnitt einer kolbenbetriebenen Pipette, dessen Querschnitt A-A mit dem Querschnitt A-A des oberen Abschnittes zusammenfällt.

Further details and advantages of the subject matter of the invention will become apparent from the following description of the accompanying drawings. In the drawings show:

Fig. 1

Co-operation of pipette tip and piston stroke pipette in a diagram with the difference of (indicated) liquid volume and piston stroke on the ordinate and the piston stroke on the abscissa;

Fig. 2

Pipette tips P ₁ , P ₂ and P ₃ to the curves A, B and C in Fig. 1;

Fig. 3

a pipette tip in a greatly enlarged, four-fold broken longitudinal section;

Fig. 4

an upper portion of a piston-operated pipette; and

Fig. 5

a lower portion of a piston operated pipette whose cross section AA coincides with the cross section AA of the upper portion.

The pipette according to FIGS. 4 and 5 is disclosed in the applicant's US-A-5511433. This disclosure is referred to. The pipette has a housing 1. At the top of the housing is a Actuating knob 2 attached, which is movable against the spring 14. The knob 2 is connected to a piston rod 7, which extends through a spindle 6. As shown in Fig. 5, the lower end of the rod 7 enters Connecting piece 8, which has a support 9 for a piston 10. A spring 18 presses a lower sleeve 19th for guiding the piston 9, 10 against an inner conical surface 21 of the housing 1. The sleeve 19 is supported on the Housing via a resilient sealing ring 20 from.

When the button 2 is pressed down vertically, the piston 9, 10 is pressed into the cylinder chamber 11, wherein he presses air from the cylinder chamber 11 through the tube 12. At the lower end of the tube 12 is a attachable Attached pipette tip, which is shown in Fig. 3 on an enlarged scale. The pipette tip is on a conical Seat attached to the lower end of the tube 12 in a manner not shown. The cylinder chamber 11, the tube 12 and the pipette tip define the dead volume of the pipette.

When pressing the button 2 vertically down the piston 9, 10 is moved until it is against the upper end of the tube 12, which is held by a stationary part in the cylinder chamber. This forms one lower stop for the piston movement.

The piston rod 7 is provided with a flange 22 on which the spindle 6 in the initial position of the rod. 7 and the piston 9, 10 rests. This flange 22 defines the upper stop for the piston 9, 10. The upper stop is adjustable as follows: The knob 2 is connected to a downwardly extending sleeve 5, which is rotatable in the housing is held. The spindle 6, which surrounds the rod 7, is rotatably connected to the rotatable sleeve 5. The outer Thread of the spindle 6 cooperates with an internal thread of a part 36 which is fixed in the housing 1. When the knob 2 is rotated, the spindle 6 is screwed into or out of the threaded portion 36. The spindle 7 is thereby adjusted axially, whereby the setting of the stop for the flange 22 of the rod 7 is set which becomes effective upon return of the piston 9, 10 to an initial position when the button 2 is released and is reset by the spring 14. The stroke of the piston is determined by the upper stop of the piston rod in limited to its starting position.

If the spindle 6 is manually rotated by means of the knob 2, a wheel 50 is adjusted via a worm gear. For this, the sleeve 5 has outside a toothing 52. The rotation of the wheel 50 is indicated by a scale, the through a window 53 is visible. The scale is calibrated to adjust the top stop so that one desired amount of liquid is sucked into the pipette tip, attached to the lower end of the tube 12 is.

As already mentioned, FIGS. 4 and 5 show part of the dead volume 11 and 12 as well as the adjusting means and Display means 51 for setting and displaying the desired amount of liquid.

In Fig. 1, the curves A ₁ and A _{2 represent} the behavior of cylindrical pipette tips P ₁ according to Fig. 2. In these pipettes, the negative volume error - ΔV increases linearly with increasing piston stroke. The curve A ₁ corresponds to a pipette tip with a smaller and the curve A _{2 of} a pipette tip with a larger cross-section.

The volume error - ΔV of the pipette tip is compensated by tuning the piston stroke pipette. Namely, by adjusting the pipette correction factor, the difference between the indicated liquid volume V _Anz. and the piston stroke V _piston in response to the piston stroke just chosen so that the indicated liquid volume V _no. the recorded liquid volume V _liquid. equivalent. Here the piston stroke pipette with the curve K ₁ and the larger pipette correction factor a of the pipette tip with the curve A ₁ and the piston stroke pipette with the curves K _{2 of} the pipette tip with the curve A _{2 are} assigned. The diagram illustrates that for each piston stroke V _piston the difference between the indicated liquid volume V no _. and the received liquid volume V _liquid. is equal to zero.

The curve B in FIG. 1 is assigned to the pipette tip P ₂ of FIG. 2. This has adjacent to the tip opening a thin suction tube, which initially causes a small piston stroke a relatively high increase in the liquid column. As a result, in this initial region, the negative volume error - ΔV is relatively high. If the liquid column fills the suction tube, a slight increase in the negative volume error is observed when rising into the adjacent cylindrical working volume.

The curve C in Fig. 1 corresponds to the pipette tip P ₃ of Fig. 2. This has adjacent to a thin suction tube a conical transition region to a cylindrical working volume. Thus, in FIG. 1, adjacent to the steep initial section, there is an area of attenuated slope of the volume error for the transition region, which eventually terminates in a region of weak slope for the cylindrical working section.

The curve C corresponds to a Kolbenhubpipette, in which the difference of the indicated liquid volume V _no. for the piston stroke V _{piston is represented} over the piston stroke through the curve K ₃ . This has on the ordinate a zero offset NV, ie the piston in the starting position is already a liquid volume V _Anz. displayed. The zero offset is chosen so that in a piston stroke V _piston , which causes an increase of the liquids in the working range of the pipette tip, just a compensation of the volume error is achieved. The system thus has an error curve F, which falls from the maximum initial value to a value zero, which is reached when the liquid column rises to the working range.

The pipette tip in Fig. 3 has at the bottom a tip opening 1 'for liquid passage, to which a thin Suction tube 2 'connects. The suction tube 2 'opens into a transition cone 3', the other with an extended work area 4 'communicates. The work area 4 'opens at the top in a Aufsteckkonus 5' with a plug-in opening 6 'for a Kolbenhubpipette with adjustable piston stroke.

In the range adjustable amounts of liquid, the liquid rises through the tip opening 1 and the Suction tube 2 and the transition cone 3 always up in the work area 4 up. To over the entire adjustment range an approximately constant absolute accuracy deviation of the amount of liquid absorbed by the To realize the indicated amount of liquid, the pipette tip in the working area 4 inside is approximately circular cylindrical shaped.

For better mold release in injection molding is both in the area of the suction tube 2, as well as in the work area 4 a slight taper provided, which the desired accuracy deviation practically not impaired. The transition cone has a clear cone angle, which helps avoid a fountain effect about 7 °.

A typical pipette tip has a total length of about 100 mm, of which about 25 mm on the suction tube 2 omitted. The diameter increases from about 0.5 mm in the tip opening 1 to about 3 mm at the beginning of the working area 4 on.

Claims (12)

1. A method for correcting the volume error ΔV in the course of designing a pipetting system comprising a plunger-operated pipette and a pipette tip adapted to be slipped onto top thereof wherein the plunger-operated pipette has adjusting devices for varying the piston stroke and display devices for the respective liquid volume pipetted and the relation of the piston stroke to the liquid volume displayed is determined by a pipette correction factor a, the pipette tip has a tip opening, a volume range coupled thereto for the reception of adjustable liquid volumes, and a slip-on opening coupled thereto for the plunger-operated pipette and a dead volume V_t is formed between the tip opening and the piston, wherein the volume error ΔV requiring correction results from the fact that the dead volume V_t between the liquid level and the piston of the pipette is increased because of the weight of the head of liquid in the pipette tip, wherein the correction of the volume error ΔV is performed by constructional agreement between the dead volume V_ts of the pipette correction factor and the contour of the cross-section Q of the pipette tip, which has a cylindrical working range in which the liquid level rises within the entire range of adjustment of the accommodatable liquid volumes, in such a way that the liquid volume A_Anz displayed by the display devices matches the liquid volume received V_Flüss or a predetermined deviation.
2. The method as claimed in claim 1, characterized in that the pipette correction factor, the contour of the inner cross-section, and the dead volume are harmonized with a zero shift of the liquid volume displayed with respect to the piston stroke.
3. The method as claimed in claim 1 or 2, characterized in that the contour of the inner cross-section along the tip is harmonized with predetermined values of the pipette correction factor, the contour of the inner cross-section, and the dead volume, if required for the latter.
4. The method as claimed in any one of claims 1 to 3, characterized in that the pipette correction factor, the contour of the inner cross-section, the dead volume, and the dead volume, if the latter is required, are harmonized with each other while taking into account the following interconnections:

   

   or

   R_rel. = R_abs./V_Flüss. = Relative deviation from correctness

   with R_abs. = V_Flüss. - V_Anz. = Absolute deviation from correctness

   V _Flüss. =

   

   = Liquid volume received

   Q(y) = Cross-section at a distance y from the tip opening

   V_Anz. = Liquid volume displayed

   ρ = Density of the liquid requiring pipetting

   g = Gravitational acceleration

   h = Height of the head of liquid in the pipette tip

   V_t = Dead volume

   P_o = Ambient pressure

   a = Pipette correction factor

   B = Zero shift.
5. The method as claimed in any one of claims 1 to 4, characterized in that a pipette tip is used in which the working range of the pipette tip has arranged in front thereof a gradually widening transition range.
6. The method as claimed in claim 5, characterized in that the transition range is determined by an angle which is as small as possible.
7. The method as claimed in any one of claims 1 to 6, characterized in that a pipette tip is used in which there is a thin suction tube adjacent to the tip opening.
8. The method as claimed in any one of claims 1 to 7, characterized in that the pipette correction factor is adjusted.
9. The method as claimed in any one of claims 1 to 8, characterized in that the zero shift of the display is adjusted.
10. The method as claimed in any one of claims 1 to 9, characterized in that the dead volume is adjusted.
11. The method as claimed in claim 10, characterized in that the dead volume is adjusted by exchanging pipette tips and/or via connecting pieces between the plunger-operated pipette and the pipette tip.
12. The method as claimed in any one of claims 1 to 11, characterized in that the dead volume is adjusted by variably setting the lower piston stroke stop.

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