Quantifying Pipette Variability due to Humidity

Laboratory error can arise from a number of sources ranging from instrumentation malfunction to sample contamination and even operator error. The quality of laboratory data is dependent on the accuracy of the tools that are used to generate that information.

One of the most commonly used laboratory tools is the pipette, and while it is a fraction of the cost or sophistication of a mass spectrometer or an analytical balance, its performance is a critical factor in laboratory results. Like other laboratory instrumentation, numerous factors affect a pipette’s accuracy and precision, including operator technique, mechanical failure, and environmental conditions in a laboratory.

To correct for pipetting variability, the source of error must be identified, understood, and addressed. For example, liquid-handling error caused by an internal device failure will require maintenance, while operator-induced error requires technique training for correction.

Understanding how environmental conditions affect the performance of pipettes is the purpose of Artel’s Extreme Pipetting Expedition, a multiphase scientific study. The team visits locations that are reflective of common laboratory environmental conditions to quantify resulting performance variability. In the first three missions, experiments were conducted to explore how barometric pressure, dry heat, and pipetting hot and cold liquids affect liquid-handling accuracy and precision (See sidebar below for more information).

This article will discuss Mission #4, which visited the rainforest of Olympic National Park in Washington to test how humidity affects pipetted volumes. Understanding how pipettes perform in high humidity is critical for laboratory data integrity, because humidity levels can vary across laboratories and even within a laboratory itself.

According to regulatory standards, pipette calibration laboratories must keep their relative humidity at greater than 50%. Working laboratories are often significantly drier, though. By visiting Olympic National Park, Artel sought to determine how pipettes behave in a humid environment and whether varying humidity levels affect pipetting accuracy and precision.

Tools and Technique

The team conducted four experiments and in each, used a number of variable-volume pipettes from leading manufacturers that dispense a maximum of 1,000, 200, 20, and 2 µL. During the experiment, each pipette was used to dispense 10 data points, each at its maximum and minimum volumes.

Volume accuracy and precision of the pipettes were tested in the extreme environment using the company’s PCS® Pipette Calibration System. The PCS is based on ratiometric photometry, which measures the absorbance of light of proprietary reagents at two distinct wavelengths to verify volume. The change in the absorbance at each wavelength is measured to calculate the actual volume pipetted. The system automatically compares actual dispensed volumes to desired target volumes and quantifies any resulting error.

Understanding the Effect

In an environment such as the Olympic National Park, both temperature and humidity must be evaluated to truly understand resulting volume variability.

In dry environments, evaporation occurs within pipette tips, leading to underdelivery of aqueous solutions. In an environment with constant relative humidity, greater evaporation occurs the warmer an environment is. The rate of evaporation is proportional to the evaporation potential, which is the difference between the partial pressure of water in air at saturation conditions and the actual partial pressure of water in air at ambient conditions. Understanding an environment’s evaporation potential is the first step in studying how humid conditions affect pipetted volumes.

To mimic pipette calibration laboratory conditions of 60% relative humidity and 20°C, Mission #4 was set for late September. In the morning of the first testing day, the humidity in the rainforest was above 60%, but the temperature was only 14°C. To understand pipette performance in the required temperature, the Artel team tested pipettes indoors at a hotel near the park. Pipettes performed accurately and precisely in this environment (Figures 1 and 2).

The next day, the team made its way to La Push First Beach, one of the beaches in Olympic National Park. Due to generator malfunctions, however, they were unable to run testing equipment. After finding a makeshift solution—a car battery—the team set up the equipment in the park at Rialto Beach. At 14°C and 74% relative humidity, the site had a very low evaporation potential, and the pipettes performed well (Figures 1 and 2).

Comparing and Correcting

Although pipettes demonstrated superb performance at Olympic National Park, most laboratories do not have similar environmental conditions. Except for pipette calibration facilities, laboratories typically operate at 15–40% relative humidity.

To determine if significant variation in pipette performance exists between the humid environment and a typical laboratory, Artel tested pipette performance at two drier facilities: lab A with 22% relative humidity and 21°C and lab B at 40% relative humidity and 22°C.

Pipette performance degraded in drier laboratory environments with a statistically significant error in lab A. The data shows that drier laboratories are more prone to liquid-handling error and that pipettes calibrated off-site in a controlled facility may underdeliver when used in normal, working laboratory conditions.

Previous Extreme-Pipetting Expeditions

MISSION 1: Mount Washington For the expedition’s first mission, the team made its way up to Mount Washington, the highest peak in the White Mountains of New Hampshire, to test the effects of altitude (low barometric pressure) on pipetted volumes. Experiments were conducted at 1,917 meters (6,288 feet) and at a barometric pressure of 805 millibar, well below the sea-level average of 1,013 millibar. The team found that at this altitude, air-displacement pipettes underdelivered by 1 to 10% due to the lower density of air at higher altitudes. When using air-displacement pipettes, if air is less dense, less liquid is aspirated into the pipette tip and subsequently dispensed, resulting in underdelivery and possible test failure. Laboratories can compensate for the repeatable volume variation caused by barometric pressure by adjusting the internal mechanism of the pipette or by adjusting the delivery setting. MISSION 2: Yellowstone National Park At Yellowstone National Park, pipettes experienced volume variation when dispensing fluids at temperatures different than the pipette itself with underdelivery of warm liquids and overdelivery of cold liquids. The error was especially significant when handling small liquid quantities. Error was present but lessened when working with larger liquid volumes. Pipette calibration regulations stipulate stringent control of temperature during calibration (20 ± 0.5°C) and that all materials including pipettes, tips, and liquids be thermally equilibrated prior to calibration. Many common assay protocols, however, require the dispensing of warm or cold reagents such as in tissue culture applications, which employ reagents at 37°C, assays with nucleic acid-based reagents at 4°C or lower, and PCR samples transferred at 60°C or warmer. MISSION 3: Death Valley National Park Artel went to Death Valley National Park to test the effect of dry and hot environmental conditions on pipetted volumes. Many technicians encounter dry laboratory conditions due to the proximity of ovens, incubators, freezers, and air-conditioning and heating systems. At Death Valley, the team found that pipettes underdeliver by up to 35% in dry and hot environments. Prewetting pipette tips did mitigate some volume variation, but underdelivery still persisted. The pipettes were also found to operate outside of manufacturers’ specifications in most instances. As in previous missions, error caused by dry heat was greater when pipettes were set to their minimum volumes than when at maximum volumes.

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Doreen Rumery is lab technical manager and QC manager at Artel. Web: www.artel-usa.com. E-mail: drumery@artel-usa.com.