Fast Gravimetric Testing of Multichannel Pipets

Piston pipets with air cushions have long been the standard equipment in many laboratories, especially in medical research and the life sciences for the transfer of liquids in the microliter range. The ease of handling of these small volumes made possible the development of many methods in the life sciences, chemical analysis, and clinical chemistry. In comparison to glass pipets, piston pipets offer clear advantages. They enable very rational and efficient operation and consequently much higher throughput.

When using piston pipets with the adaptable one-way plastic tip, the dispensed medium only comes into contact with the tip because of the air cushion above it. Therefore, pipet contamination (e.g., with radioactive or infectious material) is avoided. Cleaning after use is not necessary as it is with glass pipets.

Multichannel pipets with 4, 8, 12, or 16 channels permit even faster and more efficient operation. In particular, the use of multichannel pipets in combination with multiple-well plates significantly reduces the amount of pipetting processes required, and allows considerably faster and more efficient use.

Over time, changes such as corrosion of the piston rods or drying of the piston seals may occur. This has a significant impact on measurement accuracy. The higher the number of channels in the multichannel pipets, the higher is the probability of a defect in one of the channels. Because these alterations are not easily recognizable, it is extremely important to check the multichannel pipets at regular intervals.

It is for this purpose that a series of rules and regulations exists. According to ISO 9000 and the GLP regulations,1 all liquid handling instruments such as piston pipets must be tested, cleaned, serviced, and calibrated regularly. For medical analysis, only instruments with a certificate of conformity may be used for quantitative measurements. Pipet manufacturers are required to test their products before they leave the factory. Certified or accredited laboratories that use pipets must test them or have them tested regularly, usually at intervals of 6 or 12 months, depending on usage. This entails considerable time and cost for large laboratories that commonly use many pipets.

The requirements for measurement accuracy (maximum permissible systematic errors and random errors) for single-channel and multichannel pipets are described in ISO 8655-2,2 the successor to DIN 12650.

The uncertainty of measurement, μ, is given by the following equation:

μ = # es # + 2s                     (1)

Where μ is the uncertainty of the measurement; # es # is the systematic error; and s is the random error (repeatability standard deviation).

The systematic error of piston pipets is the deviation of the nominal or selected partial volume from the mean value of 10 measurements. The random error of piston pipets is the repeatability standard deviation of the 10 measurements.

The test is primarily carried out gravimetrically according to ISO 8655-6,3 since this is the reference method. The test procedure required by ISO 8655 is complex: The piston pipet must be conditioned first, i.e., the tip must be premoistened five times to saturate the air cushion in the pipet (dead volume) with moisture.4–6 Then, the pipet is filled by pushing the piston to the first stop, dipping the tip into water, and slowly sliding back the piston. The content of the tip is emptied into a glass tube and immediately weighed. To avoid evaporation of the water during weighing, sealed weighing vessels must be used. Alternatively, evaporation can be greatly reduced by using evaporation traps (areas with very high humidity). Correction by calculation is also possible.5,6

According to ISO 8655-6, the tips may be used only once for the volume check. A new tip must be used for each measurement. Since the pipet has already been conditioned, a single premoistening following tip exchange is sufficient. In total, this measurement, including tip exchange and premoistening, is carried out 10 times.

Using single-channel piston pipets with fixed volumes, these 10 weighings can be done in a few minutes, whereas testing multichannel pipets requires considerably more time. At 10 weighings per channel for testing, the nominal volume of a 12-channel pipet, 120 weighings are necessary. For piston pipets with variable volume, which is always the case with multichannel pipets, the nominal volume, 50% of the nominal volume, and the lower limit of the useful volume range or 10% of the nominal volume (whichever is greater) must be tested. Thus, 360 weighings are necessary in all.

Compared with the testing of a single-channel pipet with a fixed volume, the time to test a 12-channel pipet is 36× greater. Thus, the testing of only one multichannel pipet typically takes 2–3 hr.

Due to the importance of multichannel pipets in the medical sector as well areas such as the life sciences, the German Standards Committee (DIN) for Laboratory Instruments suggested a research project, the objective of which was the development of a method for testing multichannel piston pipets that is fast and in conformance with the norm.

Semiautomatic gravimetric test unit

Figure 1 - Overall view of SpeedCal gravimetric testing unit.

Figure 2 - Detailed view of testing unit. Liquid is pipetted into the filling tubes of the 12 weighing vessels, enclosed by the evaporation trap.

At the onset of the research project, different solutions were analyzed with regard to time. The greatest time savings occurred when pipetting with all 12 channels simultaneously and immediately weighing the dispensed fluid volume simultaneously. For this purpose, a multichannel weighing instrument specifically for pipet testing had to be developed. The SpeedCal test instrument, shown in Figure 1 (produced by Fraunhofer-Institute for Silicate Research, Wertheim, Germany, in cooperation with Sartorius AG, Göttingen, Germany) reduces the cost of pipet testing while enabling easy, fast, and reliable testing in accordance with applicable standards. The instrument can test up to 12 pipet channels simultaneously. The weighing vessels are enclosed in an evaporation trap (Figure 2) to minimize measurement error due to evaporation. Each chamber has a volume of 12 mL; thus several pipets can be tested without having to empty the chambers between tests. The multichannel weighing instrument is automatically tared after each measurement. Once the weighing vessels are filled up, the operator can continue testing without interruption by simply suctioning off the liquid with a special device. With a 12-channel pipet, for example, simultaneous testing of channels reduces the actual testing time for 360 measurements to 10–12 min, depending on the operator's level of pipetting experience. The control of the test procedure and evaluation of results are carried out by a computer connected with SpeedCal. To convert the weighing result to the volume, the air pressure, air temperature, and water temperature must be entered prior to testing. Following each pipetting, at the touch of a button the measured weights of the delivered volumes are read after the settling of the weighing cells (approx. 10 sec), and are automatically converted into the corresponding volume.

Figure 3 - Original printout of a test record using SpeedCal. Testing of a 12-channel pipet, nominal volume 300 μL, setting 150 μL. All 120 measured values are shown (in μL). In addition to the mean value and standard deviation taken from all measurements, the mean value and standard deviation of each channel and each repeat measurement are also shown. It took about 4 min to perform these 120 measurements, including the evaluation and printout. The same period of time is required for testing at the other two volume settings; thus it takes about 12 min to complete the entire test series of a 12-channel pipet (equal to 360 measurements).

A record of the measurements (Figure 3) is printed automatically. In addition to the individual values, this test record includes mean values and standard deviations for each measurement, i.e., the mean and standard deviation among the 12 channels, as well as for each channel—in other words, the mean and standard deviation over the entire test series. Furthermore, the record shows the prevailing ambient conditions, such as temperature, air pressure, density of air and water, and the resulting conversion factor Z for deriving the volume from the gravimetric tests. Gravimetric testing with SpeedCal is fully compliant with ISO 8655 and DIN 12650, and delivers results identical to those obtained with conventional gravimetric methods (Figure 4).

Figure 4 - Comparison of the results achieved with SpeedCal and the traditional gravimetric method. A 12-channel pipet with a nominal volume of 300 μL was tested at a setting of 150 μL. The diagram shows the systematic error and two times the random error, as well as the sums of these values, which represent the uncertainty of measurement for the pipet.

With SpeedCal, the time needed to test single-channel pipets is reduced as well. The 12 vessels allow the testing of single-channel pipets without having to wait until the balances are stationary. This takes up to 50% less time than is required for conventional testing of single-channel pipets.

With its significantly faster operation and automatic documentation features, SpeedCal is well suited not only for calibration laboratories and pipet manufacturers, but also for any laboratory that tests large numbers of multichannel and single-channel pipets on a regular basis.

References

  1. OECD principles of good laboratory practice. OECD guidelines for testing of chemicals. Paris, France: OECD, 1981.
  2. Norm ISO 8655-2, 2002. Piston-operated volumetric apparatus. Part 2: piston pipettes.
  3. Norm ISO 8655-6, 2002. Piston-operated volumetric apparatus. Part 6: gravimetric methods for the determination of measurement error.
  4. Zeman GH, Mathewson NS. Necessity of prerinsing disposable polypropylene pipet tips. Clin Chem 1974; 20:487–98.
  5. Michel F, Sommer K, Spieweck F. Investigations for the determination of uncertainty in the measurement of piston pipets with volumes from 1 μL to 50 μL. PTB Mitteilungen 1995; 105:437–44.
  6. Lochner KH, Ballweg T, Fahrenkrog HH. Investigations for the accuracy of piston pipets with air cushion. J Lab Med 1996; 20:430–40.

Dr. Lochner is Senior Research Scientist, Fraunhofer-Institute for Silicate Research, Bronnbach Branch, Bronnbach 28, 97877 Wertheim, Germany; tel.: +49 9342 9221 701; fax: +49 9342 9221 799; e-mail: karlheinz.lochner@isc.fhg.de. The author is indebted to Dr. Burkhard Winter and Mr. Wolfhard Gögge of the German DIN Standards Committee for Laboratory Instruments for their valuable input, and Mr. Wilfried Langner and Mr. Jörg Barankewitz of Sartorius AG (Göttingen, Germany) for their support and helpful suggestions during the development of the SpeedCal semiautomatic gravimetric testing unit.

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