Life Science
by A. Björn Carle, Doreen Rumery, and George Rodrigues
very day, air-displacement pipets are used to quantitatively dis- pense sample and reagent aliquots for reaction, routine analyses, and specialized tests. Since concentrations of biological and chemical components in the prepared samples for analyses and as- says are volume-dependent, incorrectly performed pipetting steps will directly impact the transferred volumes, and hence, the test results. The design and construction of piston-operated air-displacement pipets render their performance susceptible to the pipetting technique and skills used by the operator of such devices. The pipet operator usually has the ability to mitigate the influence of most parameters by using the appropriate technique, as well as by choosing the appropriate pipet size and type of pipet tips.
Influence of the air cushion on pipet performance Piston-operated air-displacement pipets use an air cushion to couple
the pipet’s piston to the aspirated liquid inside of the pipet tip. This air cushion, often referred to as captive air volume or dead air volume, is trapped within the pipet as soon as the tip is immersed in the sample solution. This captive air volume closely obeys the Ideal Gas Law (Pa pressure of the trapped gas, Va its volume, na the temperature of the gas):
Pa Va = na R Ta
The Ideal Gas Law allows one to estimate the effects that temperature and, by extension, evaporation, and the ratio of captive air volume to the pipet’s set volume will have on the actually aspirated and delivered vol- ume of a pipetting cycle.1
is the the number of moles, and Ta
Best Practices for the Use of Micropipets E
Figure 1 – Volume deliveries of a 20-μL pipet. Each delivery used a new tip and was either prewet three times or not prewet prior to sample delivery.
Temperature disequilibrium For most accurate pipetting results, it is recommended that the pipet, the pipet tip, and the sample solution have been equilibrated for at least 2 hr and are within 0.5 °C of ambient temperature.3
Many samples, however,
must be handled at specific high or low temperatures, and pipetting such samples can introduce significant errors in the delivered volume due to the expansion or contraction of the captive air volume and evaporation. Studies of this effect have been reported previously.4
The following techniques studied here directly
influence the captive air volume: prewetting of pipet tips, temperature disequilibrium, hand warming, and immersion depth of pipet tip.
Since the total volume of the air cushion can vary widely depending on the type of pipet, the tip type and size, and the amount of the aspirated liquid aliquot, this study evaluated two different scenarios: one set of ex- periments was conducted with a 20-μL pipet set at 20 μL, and the other experiments with a 100-μL pipet set at 20 μL.
Prewetting of pipet tips Sample solution in the pipet tip is susceptible to evaporation into the air cushion during and after aspiration. The evaporative loss of sample solution is dependent on the humidity of the captive air space, as well as the temperature of the sample solution. Repeated aspiration/dispense cycles will increase the humidity of the air in the pipet tip and shaft. Figure 1 shows the dispensed volumes of a 20-μL pipet set to 20 μL and used in a noncontrolled laboratory (30% relative humidity). Each dispense was performed with a new tip. Without prewetting the tips, the pipet dispensed on average 1.3% less volume, as compared to dispenses when the tip was prewetted three times prior to the dispense. When using pipets in particularly dry or warm environments, the error can be significantly larger without prewetting the tips.2
The present study (see experimental conditions) evaluated the use of pipet tips that had been cooled to 4 °C for 30 min prior to use. Pipetting with these cold tips led to significant underdelivery of sample with both pipets, contributing up to –1.9% relative inaccuracy (RI) and 1.2% coeffi- cient of variance (CV) to the errors. The inaccuracy and imprecision results for both pipets and all tested scenarios described here are graphed in Figures 2–5. Experimental conditions are shown in Table 1.
Heat transfer/hand warming Handling a pipet for prolonged periods of time will cause the barrel of the pipet to warm, leading to an expansion of the captive air volume, ultimately impacting the accuracy and precision. Progressive warming of the pipet’s barrel through heat transfer from the hand manifests itself by a trend toward smaller delivered volumes, and led to –1.1% RI and 0.8% CV in this study.
Immersion depth of the pipet tip Immersing the pipet tip to the proper depth during aspiration of the sample is important. Pipet calibration standards like ASTM E1154 recom- mend5
an immersion depth of 2–3 mm for pipet volumes of 1–100 μL,
2–4 mm for 101–1000 μL, and 3–6 mm for volumes larger than 1 mL. In this study we evaluated immersion depths of 1 mm and 8 mm. A shal- low immersion depth increases the risk of aspirating small amounts of air, while immersing tips too deeply increases the risk of carrying over droplets on the outside of the tip, and/or forcing more sample in the tip due to increased hydrostatic pressure on the outside of the tip. Either case leads to a significantly increased imprecision (up to 2.2% CV) of the delivered volumes.
AMERICAN LABORATORY • 14 • JUNE/JULY 2014
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