FI98604C - Method for Improving the Pipetting Accuracy of a Volumetric Pipette and a Pipette Providing Improved Pipetting Accuracy - Google Patents

Description

98604

Method for Improving the Pipetting Accuracy of a Volumetric Pipette and a Pipette Providing Improved Pipetting Accuracy 5

The invention relates to a method for improving the accuracy of pipetting. The method is preferably used in conjunction with an electronic microprocessor-controlled pipetting device described in U.S. Pat.

10 5,343,769 (Suovaniemi & Ekholm) as well as in connection with mechanical pipettes. The invention also relates to a new pipette with improved pipetting accuracy.

Laboratory analyzes most of the liquid in the form of 15 pipetting and dispensing of samples and reagents are the most difficult step to achieve e.g. less than half a percent accuracy for a match so as reproducibility. Pipetting accuracy with the more advanced technique has most commonly been on the order of one microliter (up to 5-20 10% inaccuracy, especially at the smallest volumes), if not worse (Rodgerson et al., Clin. Chem. 20/1, 43-50, 1974 and Pardue et al., Clin Chem.

20/8, 1028-1042, 1974). Applying today's modern technologies, the accuracy of both mechanical and electronic pipettes varies in the range of 0.1 to 5% «(Suovaniemi: dissertation, ISBN 952-90-5248-0, University of Helsinki 1994).

• · · • · ♦ · 98604 2 keen; e.g. the 1000 μ1: η setting can return the tip to its original shape with up to 1030 - 1040 μ1: η amounts of liquid to the pipette tip, which error can be eliminated e.g. by using vertically packed tips or by handling the tips very gently without changing their shape when pipetting. The use of an electronic pipette eliminates human-induced pipetting errors as well as the automatic calibration of the electronic pipette whenever it is used (Hattori, 10 Super Pipetter Seminar: Standardized Risk Factor and Support to Validation, Biohit, 1994). By improving these factors, in most cases only 0.1 to 0.5% more accurate pipetting results are achieved. It has also been found that even liquids of different viscosities can be pipetted with an accuracy of less than 1% with devices based on current technology.

No attention has yet been paid to the effect of pipetting accuracy on the pipetting tip, the pipette and its tip, and the ambient temperature and / or pressure in the air space between the pipette tip and the plunger. The viscosity and density of the liquid to be pipetted have also not received much attention.

• v 25 ·· ·: It has now been found according to the invention that considerably better pipetting results can be obtained by the method, the features of which are set out in the appended claims.

More specifically, the said better pipetting accuracy;. ·. 30 to six is achieved by considering the volume control of the pipette, especially the liquid to be pipetted, but • · · *. also the tip of the pipette and the air it contains, • ‘·· the temperature of the pipette itself and / or the ambient temperature. The temperature can be taken into account by correcting the setpoint of the pipette according to the measured temperature 35, or by taking into account the difference between the two temperatures, using a correction value or a correction factor of 98604 3 to adjust the setpoint. , on the basis of which the microprocessor, using the parameters in its memory, makes the necessary corrections to the volume to be pipetted 5 to correspond to the set value of the pipette for each volume.

According to the invention, it is also possible to take into account, in addition to the above-mentioned temperatures, the pressure in the air space between the pipette tip and the piston 10 and also to take it into account to correct the volume given by the pipette. Likewise, correction factors can be used to correct pipetting errors due to the viscosity and density of the liquids to be pipetted.

15

To illustrate the invention, reference is now made to the accompanying Figure 1, which shows the pipetting results when pipetting a liquid with a pipette (Biohit Proline) setpoint of 1000 μΐ when the temperatures of the liquid to be pipetted and the pipe 20 itself vary (4.6, 22.2 and 28.3 ° C, respectively). ). It can be stated that the set value of 1000 μΐ reaches the corresponding volume when the pipetting liquid, ··· the tip of the pipette and the pipette itself are, for example, at room temperature.

: lassa (22.2 ° C). These are the only conditions in which NCCLS

• 25 (National Committee for Clinical Laboratory Standards: • · · j V Proposed Guideline, Determining Performance of Volumetric j:: Equipment, Vol 4, No 6) provides guidance and on which the quality assurance of pipette manufacturers is based. In other respects, the manufacturer is left without instructions. The NCCLS does not comment on:. 30 what happens to the test results of the pipette user and possibly • · · based on these erroneous results- • · · *. NCCSL if the user of a 'high and uniform' pipette deviates from the same, constant temperature with respect to the pipette, its tip or the liquid to be pipetted.

Figure 1 shows that when the liquid to be pipetted, the pipette itself and the ambient temperature are the same (4.6, 22.2;. 35 98604 4 or 28.3 ° C), a set value of 1000 μ1: η achieves a corresponding volume in each pipetting. temperature.

This is because during the pipetting step, when the liquid is sucked into the tip of the pipette, there is no change in the temperature of the pipette, i.e. a corresponding change in pressure and volume, and with it a change in the amount of liquid sucked into the tip.

This volume can be calculated from Gay-Lussac's law, V, = VQ x 10 T / T0, according to which the volume of air at and above the pipette tip during the phase of aspirating the liquid into the pipette tip, Vt, is the volume of liquid taken at t directly proportional to the corresponding absolute temperature obtained in degrees Kelvin-15 by adding a constant 273 to degrees Celsius and denoted by T. This means that the liquid to be pipetted into the pipette tip at + 4.6 ° C does not change the temperature of the pipette tip and the 4.6 ° C air in it, i.e. T and T0 are the same (277.6 ° K) before aspirating the liquid 20 into and during the pipette tip.

On the other hand, when pipetting 1000 μ1: η with a pipette at room temperature (22.2 ° C) and a tip at 4.6 ° C 'l', 'water of the same temperature (4.6 ° C) is obtained; 25 pipetting result 945 μΐ. When the temperature changes during the • · ·: · * phase when the liquid is drawn into the pipette tip (the air space between the liquid and the pipette piston is closed) and • · · x .; · The volume to be pipetted according to the setpoint is taken into account

In the formula according to Gay-Lussac's law, so that 1000: 30 μ1: η of the liquid space corresponds to a gas space of the same size, we obtain • «· ·: * · *: volume by calculating 1000 μΐ x 277.6 / 295.2 = 940 μΐ m, ie slightly less than that obtained by pipetting, 945 μΐ. The difference •, is due to the fact that the cold ‘·’ (4.6 ° C) liquid surface being sucked into the pipette tip by the air 35 above it in the closed space between the liquid surface and the plunger; in the short part, it decreases and at the same time shrinks (pressure decreases) and thus also affects the air displacement from the tip of the pipette. As a result of this pressure drop, a little more liquid is brought to the tip. Ko. the significance of the pressure drop in the difference between the volume of pipetted liquid and the corresponding calculated value is estimated to be much smaller than the tendency of the air temperature in the liquid state of the tip itself to equalize under the influence of the tip and the surrounding higher temperature. This increases the volume of the gas space that becomes a closed liquid space in the pipetting situation and the deviation from the calculated volume value.

At a pipette and tip temperature of 22.2 ° C and a pipetting liquid temperature of 28.3 ° C with a setting of 1000 μ1: η, the pipetting result is 1018 μΐ, which almost corresponds to the legally calculated value of 1020 μΐ for Gay-Lus-15. Since the temperature-lagrad gradient is not very large, only 6.1 ° C, according to this experiment, the pipetting result corresponds almost to the calculated value. The volume obtained by pipetting (1018 μΐ) is slightly less than the calculated volume (1020 μΐ) because the aspirated liquid at the tip of the pipette, 20 warmer than it and the pipette air space, in the enclosed air space between the pipette plunger. This in turn reduces the volume of fluid sucked to the 25 tips from the calculated value, 1020 μΐ.

• · • · f · · I · · • · # ·, * j ': In addition, experiments were performed to determine the effect of the temperature of the liquid to be pipetted, the pipette and the pipette tip on the volume of the liquid to be pipetted. The liquid to be pipetted was water, using a Biohit Proline • · · "electronic pipette. The pipette was set to 1000 μΐ, and • the corresponding pipetted volumes were weighed in mg with the Biohit i: Ohaus system. No water density correction was performed.

The results are shown in the following Table 1. In 98604 6, columns 1 and 2 correspond to an experiment in which three pipetting was performed before the pipetting values considered. In the experiment in column 3, pipetting was started directly on the balance. In the experiment in column 4, the dry tip was replaced before piping the ku-5 jacket. In the experiment in column 5, pipetting was started directly on the balance. The experiments in column 6 are a control of the measurement results in column 1.

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From column 2 of Table 1, it can be seen that when pipetting water at 8 ° C with a room temperature (26 ° C) pipette and tip, the pipetting result is 946.6 μΐ 5 (average), which is slightly higher than calculated for the reason explained above. value obtained, 940 μΐ. Of the pipetting values in column 3 under the same conditions as in the previous pipetting, the first pipetting value (1004.7 μΐ) corresponds to 10 setpoints (1000 μΐ), taking into account other pipetting errors, because the air displaced by the liquid at the pipette tip does not reach the suction water temperature. C. The amount of liquid obtained in the second and especially the third pipetting drops to the steady-state level (948 15 μΐ), the corresponding value obtained by calculating the volume of air being 940 μΐ. The same effect on the amount of liquid to be pipetted can be seen in column 4. By changing the dry tip at room temperature for each pipetting, the effect of the low temperature of the liquid on the amount of liquid to be pipetted is virtually eliminated. It can be seen from column 5 that if the temperature of the pipette tip is the same as that of the liquid to be pipetted, the pipetting results correspond to the phenomenon described above. The results in column 6 are a control for the pipetting results in column 1, which are obtained when both the liquid to be pipetted and the temperature of the pipette and tip are the same, 26 ° C.

• · I · · • · • · ·, 1J1. In the second set of experiments, water was also pipetted with a Biohit Proline electronic pipette; setpoint 1000 μΐ. Pipette-. <· Ι Weighing of 30 volumes of liquid in mg with the Biohit Ohaus system • · · “! .1; no water density correction was made.

• · · • · ·

Il · • ’·· The results obtained are shown in Table 2 below.

In the column of Table 1, pipetted 26 ° C water with a pipette at the same temperature at a pipette tip temperature of 8 ° C. In the experiment in column 2, the pipette was at 26 ° C and the tip of the pipette and the liquid to be pipetted at 9 ° C.

98604 9

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The same phenomena can be observed from Table 2 as from Table 1, except that only lowering the temperature of the pipette tip does not have a very large error-causing effect (see column 1), which is also evident from the results in column 2. Essentially, only the difference in temperature of the liquid to be pipetted from the temperature of the pipette causes the largest error in the pipetting results. It can be seen from column 3 that during the first pipetting of the first 10, it is apparent that the tip temperature has been higher than that of the liquid to be pipetted, which is proportional to the result level of column 4. The test series in column 5 corresponds to the results in the test series in column 5 of Table 1, the only difference in conditions being that the results in the previous 15 columns were measured with water at 8 ° C and the latter at 9 ° C. The results in column 6 are consistent because pipetting room temperature (26 ° C) air between each pipetting raises the air at the tip of the pipette and the air displaced by the liquid closer to room temperature, 26 ° C.

The above test results thus show that pipetting liquids at different temperatures causes considerable deviations from the pipette setpoints, up to 5-6%, 25 which error must definitely be taken into account in practical V quality laboratory work. The above ♦ ♦ · ϊ ·: test results confirm that pipetting of a pipette, i.e. usually t liquids other than room temperature · »· ·! * · *; results in the initiation of time-dependent thermal processes during pipetting by pipette manufacturers; ignore and do not inform users (cf. NCCLS 4 m Φ

Propose Guideline). The effect of these processes on the pipette »·« *. the volume of the liquid sucked into and dispensed from the tip «· • * ·· is very significant and surprising even with small temperature differences depending on their time duration and how soon the steady-state of the changes * ..., ie the constant change as a function of time, is reached . These problems occur especially in the most widely used so-called air-displacement-type 98604 12 long pipettes in which a closed air space (air column) is left between the plunger of the pipette and its liquid reservoir during pipetting and in which the temperature and pressure dependent air column (its volume 5) at the pipette tip is replaced by a liquid equivalent .

Thus, based on the above test results and considerations, it can be shown that the volume of the liquid to be pipetted at the tip of the pipette is affected by the volume, temperature and pressure of the air displaced by the liquid. Based on the above test results, when pipetting reagents and samples from an ice bath (about 4-5 ° C) with a room temperature pipette and tip, which is the most common working situation, especially in research laboratories, the pipetting results deviate by 5-6% from the desired pipette setpoint. This is an error that researchers have not taken into account when evaluating and drawing conclusions from their research results 20 quite often, considering differences of even less than one percent in their measurement results to be significant. This problem has not been taken into account because it has not been noticed.

The invention also relates to a volume-adjustable pipette, i.e. a mechanical or electric pipette, comprising a cylinder space forming a pipette air space with a fluid connection to the tip of the pipette, in the cylinder space back and forth i:: * “/ moving piston and actuators for the piston , which it * · '' pipette is characterized in that it includes means for measuring the temperature 30. A suitable means for measuring temperature • »I.i: is, for example, an NTC resistor (Ne- ·« ·: gative Temperature Coefficient resistor) acting as a temperature sensor. Such resistors are known per se and are commercially available.

35 A simple solution according to the invention is that the air space between the tip of the pipette and the plunger, preferably close to the tip, is placed in the structure of the pipette.

II

i 98604 13 NTC resistor that measures the the temperature of the room and indicates it on a display in the pipette structure, eg by means of suitable electronics. Other functions such as ambient temperature and pressure as well as time and date can also be connected to the same display. The display can also register the above airspace pressure. The pressure is measured with a suitable pressure sensor. By taking into account the pressure and / or temperature values, corresponding corrections are made to the pipette adjustment. In its simplest form. the information em.

10 of the air space temperature, preferably after the pipette has reached its steady state, for example after three pipetting. In electronic pipettes, the display may be connected to a microprocessor connected to it, the memory of which contains a correction factor corresponding to each of the above-mentioned temperature (and / or pressure) values by means of a pipette adjustment.

According to another embodiment, the NTC resistor may be located adjacent to the tip of the pipette or to the tip itself.

20

In a very simple way, the setpoint correction can be implemented by determining the correction values separately and tabulating and taking into account the said correction need, e.g. manually in mechanical pipette control, or by entering information about the correction requirement into an electronic pipette · * · * · microprocessor controlling the required correction.

% · • · »♦« • »•« · ·; *, One solution of the electric pipette is shown in the accompanying Figure I · · 2. It shows the tip part of a pipette (e.g. according to U.S. Pat. No. 5,343,769) with air space 1 • * · •; jt! the plunger 2 moves 2. In the embodiment shown, a replaceable tip 3 is mounted on the pipette, in which the liquid to be pipetted is sucked. In the embodiment shown, sensors for measuring temperature and / or pressure can be installed in the air space. According to the invention, said sensors can be suitably placed as close as possible to the tip of the pipette, such as at point 4 ', 98604 14 or at the bottom of the airspace, at point 4 ". in turn, on the basis of the input data, it controls the movement of the piston so that the amount of liquid pipetted corresponds to the set value of the pipette.

The liquid deviating from the room temperature is preferably pipetted, e.g., three times, and then the microprocessor-10 ri takes into account automatically or by a separate command. air space temperature in the pipette control at different setpoints and thus correct the pipetting results to match the pipette setpoints. Alternatively, the air space pressure in addition to temperature and take into account these values or only the pressure value to achieve pipetting values corresponding to the setpoints.

The same principles can be followed for both mechanical and electrical pipettes to make the pi-20 pipette operate at its volumetric setpoints with liquids of varying density and viscosity.

This can be accomplished by correcting the pipette setpoint with a correction factor for each fluid. In an electric pipette, this is suitably done by entering the corresponding information into the microprocessor of the pipette.

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Claims (11)

A method for improving the pipetting accuracy of a tilet volume adjustable pipette, characterized in that the setpoint of the pipette, which corresponds to the amount of liquid to be pipetted, is corrected by taking into account the sillage in temperature between the liquid to be pipetted and the temperature of the pipette. , the pipette tip or the air it contains, of the air space between the piston which controls the volume of the pipette and the liquid column containing the tip and / or the ambient temperature so that a quantity of pipetting corresponding to the set value is obtained. 2. A method according to claim 1 for improving the pipetting accuracy of an electric pipette, characterized in that a correction information corresponding to said temperature difference is measured for the pipette control, which gives the corresponding correction order for the movement of the pipette piston. 20 Method according to claim 1 or 2, characterized in that the temperature of the liquid to be pipetted is taken into account by measuring it with a probe adjacent to the pipette tip or in the tip structure, preferably an NTC sensor. 4. A method according to any one of claims 1-3, characterized in: 1 · 1: characterized in that the temperature of the air contained in the pipette tip is measured with one in the pipette structure, Preferably as close as possible to its tip position sensor, especially an NTC sensor. · · 98604 to the pipette controller. Method according to any of the preceding claims, characterized in that when adjusting the setpoint of the pipette 5, the viscosity and / or the density of the liquid to be pipetted is also taken into account by correcting the setpoint with a value relating to the liquid. ga, especially by providing the corresponding correction factor to a microprocessor in an electric pipette. 10 7. The volume adjustable pipette with a reciprocating piston in a cylinder compartment which forms the pipette's air space, as well as drive means for the cow, characterized in that the pipette contains a temperature sensor. 15 Pipette according to claim 7, characterized in that the temperature sensor is an NTC resistor. Pipette as claimed in claim 7 or 8, characterized in that the sensor is located in the air space between the cow and the tip, or near the tip. A pipette as claimed in any one of claims 7-9, further comprising a control unit for controlling electric drive means, characterized in that it has means for transmitting the measured temperature information to the control unit of the pipette. • ♦ • · • ♦ · • «j. ·. 11. A pipette according to any of claims 7-10, characterized in that it has a pressure transducer and means for transmitting the pressure information measured by the pressure transducer to the control unit. • · · • · · t »t« · · «· II