In life-science laboratories, which commonly include technologically advanced instruments, scientists often have various tools to complete everyday tasks such as liquid-handling quality assurance. Several options are available to laboratories for calibrating liquid-handling instrumentation and measuring the efficacy of liquid-handling processes. Each option has its own applications, benefits, and drawbacks. The optimal technology for a laboratory application depends on factors such as the volume of liquids to be quantified, the type of instrumentation used, and the applicable regulatory and quality standards. Also to be considered are the laboratory environment, tolerance for risk, required calibration frequency, and the demands of the laboratory's processes.
This article will compare gravimetry, fluorometry, single-dye photometry, and ratiometric photometry (common means for verifying liquid-handling instrumentation) and provide data and guidance regarding best applications of each.Gravimetry
Gravimetry has several advantages, including the wide availability of weighing devices in most laboratories. In addition, gravimetry is a well-accepted technology. It is recognized by national and international regulatory agencies, including the International Organization for Standardization (ISO), the College of American Pathologists, and ASTM International. Published standard methods of gravimetry include ASTM E1154 and ISO 8655-6 (1, 2). Gravimetric calibration can also be traced to national standards, thus facilitating regulatory compliance and standardization.
Gravimetry is frequently the method of choice for measuring device performance when handling larger volumes. For example, a 1000-μL aliquot weighs approximately 1 g and can be weighed reliably on a modern laboratory analytical balance. The current trend in laboratories toward handling small liquid volumes with automated devices illustrates one major drawback of this method: as volumes decrease, weighing becomes more challenging for several reasons.
First, measuring small liquid volumes requires specialized balances that produce measurement results to five or six decimal places on the gram scale. Such balances are delicate, require a stable platform to limit vibration, and are not as portable as the less-sensitive models used for measuring large liquid volumes. These requirements often make microgram balances unsuitable for use on the deck of automated liquid handlers. Illustrating the need for sensitivity, ISO 8655-6 requires that volumes of 10 μL or less be measured on a six-place (microgram) balance (2).
Because microgram balances take time to settle, gravimetric calibration can also be time-consuming. In addition, gravimetry is affected by various environmental conditions, including evaporation and static electricity. As volumes become smaller, these error sources become more significant.
For example, modern dispensing equipment can deliver volumes so small that they can evaporate in seconds. Obtaining adequate resolution for small volumes requires a highly sensitive balance with complicated evaporation traps, static eliminators, and vibration dampeners. Other methods for controlling for evaporation can be complicated. One method is to measure the evaporation rate and correct for the resulting volume variation. Alternatively, the humidity in the room can be increased or a draft shield built to prevent air from flowing over the testing area. These steps add time and complexity to the measurement process.