Whether manual or automated, using a pipette seems so easy—simply set the volume you’d like to pipette, then aspirate and dispense. But that simplicity can be deceptive. In addition to the many ways technique can affect manual pipetting, liquid class can affect automated liquid handling, and the chemical nature of the sample as well as environmental conditions can affect both types of instruments, there’s also normal wear and tear to factor in. And with pipetting performance often critical for assay reproducibility and validity, verifying that your pipettes are accurately and precisely transferring the desired volume within the required specifications is an essential quality assurance activity for most life sciences labs.
What’s the best way to perform volume verification checks on your liquid handlers? As with most things, that depends on your specific situation. To help you choose, we’ve put together a quick comparison table (Table 1, bottom of the page) that you can also download from here. Basically, there are four major methods—gravimetry, fluorometry, single dye photometry, and ratiometric photometry, which we discuss in more detail below.
How it works:
This method uses a balance to weigh the transferred liquid. The weight of the liquid is converted to mass, and then the mass is converted to volume based on the density of the liquid.
What it’s best for:
Gravimetry works best for measuring the performance of single-channel devices, such as manual pipettes, and when handling larger liquid volumes (~20 to 1000 μL).
Most labs already have a balance.
The technology is well-accepted and recognized by national and international regulatory agencies, including the International Organization for Standardization (ISO), the College of American Pathologists (CAP), and ASTM International.
There are published standard methods, including ASTM E1154 and ISO 8655-6.
The method can be traceable to NIST standards (if using a balance certified to NIST standards), facilitating regulatory compliance and standardization.
The method can be time-consuming, as microgram balances take time to settle. In addition, because individual wells in a multiwell plate cannot be independently assessed, gravimetric methods can only measure one channel at a time.
Gravimetry is strongly affected by environmental conditions, such as static electricity, humidity, temperature, airflow, and vibration, and these environmental effects become more pronounced as the volume being verified becomes smaller.
Verifying smaller volumes also requires more specialized balances that can produce measurement results to five or six decimal places on the gram scale. Not every lab has such specialized instruments, and controlling for environmental conditions, such as preventing evaporation of small samples or ensuring reduced static electricity in the presence of plastic pipette tips adds to the complexity of the setup.
Calibration depends on an accurate knowledge of the density of the solution being transferred. While most labs estimate the density of aqueous solutions to be 1 g/mL, the density of water, water’s actual density varies with temperature and is typically less than 1 g/mL at room temperature. Failure to correct for density errors, even when pipetting water, can lead to error in the 0.3 to 0.5% range, which is nearly as large as the acceptable error for the entire piece of equipment if accuracy is specified at better than 0.6%. And for many drug discovery and development labs, the solutions being transferred are DMSO-based, which has a density of 1.1, but is also highly hygroscopic, with the water content affecting density.
How it works:
Known amounts of a fluorescent dye are added to a solution, and the fluorescence intensity measured—fluorescence intensity is proportional to the amount of dye present, and therefore provides a measurement of the volume transferred. Precision is measured by comparing relative fluorescence intensity between different samples.
What it’s best for:
Fluorometry is best for demonstrating precision across nearly identical conditions when accuracy and traceability are not required.
Because the fluorescence signal is strong, this method can be used for verifying very small volumes, down to 5 nL.
The strength of the fluorescent signal is highly dependent on the chemical composition of the sample, i.e. solvent composition, pH, ionic strength, redox potential, time, etc., and is affected by quenching and photobleaching, which have a number of implications:
There are no commercially-available fluorometry-based calibration technologies.
How it works:
A known amount of stable, light-absorbing dye is added to a sample, and then a spectrophotometer is used to measure absorbance. The amount of light that is absorbed is proportional to the amount of dye present, enabling calculation of the sample volume.
What it’s best for:
Single dye photometry is best for measuring precision. Accuracy measurements can also be made, although their robustness is limited due to the difficulty of ensuring that the method is properly standardized and that an uncertainty analysis yields acceptable performance.
The method is less sensitive to environmental conditions than gravimetric and fluorometric calibration technologies.
Photometric dyes are typically less sensitive to the chemical composition of the sample than fluorometric dyes.
While photometric dyes do change due to temperature and pH, they tend to be more stable than fluorescent dyes, resulting in more consistent measurements.
There are commercially available single dye photometry-based technologies.
ISO 8655-7 recognizes the use of single-dye photometry for liquid handling device calibration. However, according to this standard, photometric methods should be accompanied by an analysis that describes the measurement uncertainty.
Because ISO 8655-7 requires an uncertainty analysis, the user must provide information on the accuracy of the photometer and reagents, dye instability, deviation from ideal Beer’s Law behavior and the like, which can be complex. For example, to account for the dyes as a source of error, data on the stability of the dye, either from the manufacturer or developed in-house through a stability or validation study, is important. In addition, the optical quality of the microtiter plate or cuvette used can also affect the accuracy and precision of the measurement and must also be accounted for.
The traceability of the method depends on many factors, including how carefully standardization is carried out. To obtain traceable photometric readings, a standard curve must be developed using a known liquid delivery device (calibrated pipette) or by weighing volumes. This process can be time consuming and tedious and assumes that the liquid handling device used to develop the standard curve is itself reliable.
How it works:
This method overcomes the accuracy limitations of single dye photometry resulting in a robust, easy-to-implement calibration method. With ratiometric photometry, a spectrophotometer measures the absorbance of two known dyes to determine the amount of each dye present, and the ratio of one dye to another. While the absolute absorbance value of a single dye can vary over time (e.g., due to evaporation, etc), the ratio of absorbance of two dyes is more stable (the factors that affect absorbance accuracy have a favorable covariance when measured in a ratio), enabling more robust accuracy determination.
What it’s best for:
This method can measure both accuracy and precision simultaneously across a wide volume range, and can be used to calibrate single-channel and multi-channel pipetting devices.
The primary benefit of this approach is its ability to improve the accuracy and robustness of measurement in comparison to non-ratiometric methods. Absorbance ratios can be measured more accurately than individual absolute absorbances.
Compared to gravimetry, this method offers greater speed, ease-of-use and enhanced accuracy in small-volume measurements.
Compared to fluorometry, ratiometric photometry provides measurements that are traceable to internationally recognized standards which allows for inter-laboratory comparability regardless of lab location (e.g., anywhere in the world), or elapsed time (measurements can be made days, months or years apart).
Systems based on ratiometric photometry provide information about each individual channel in multichannel devices and good reproducibility plate to plate.
There are commercially available technologies—for example, the Tecan QC Kit is a ready-to-use volume verification kit suitable for both internal and compliance uses, as well as Artel’s PCS and MVS Instruments.
Ratiometric photometry methods require use of specially formulated dyes in order to produce accurate absorbance ratios.
This technology is not always preferred when measuring only larger volumes (> 5 mL), as other technologies may produce adequate measurements more cost effectively.
Ensuring that liquid handlers—whether they’re manual or automated—are dispensing the desired volumes can be critical for diagnostics, biotech, and pharma QA. And while there are many different methods, the best one is the method that is easy-to-do, within your lab’s budget, and meets your compliance needs.
Melinda Gold, PhD is a Product Manager at Artel where she supports product development, strategic planning and uses scientific marketing to develop sales strategies and tactics to further grow product lines to satisfy customer needs. By developing applications, market and client specific solutions, Dr. Gold works to assist customers with their challenges through training and utilization of her knowledge and expertise in liquid handling quality. Dr. Gold earned her BS from Fordham University in Computer Science and her MS and PhD from St Elisabeth University of Health and Social Sciences in Bratislava, Slovak Republic in Public Health and Molecular Biology respectively. Dr. Gold has worked as a product manager in the liquid handing field for over 8 years and has a great deal of experience with liquid handling instruments and the challenges that accompany accurate and precise liquid delivery.