The accuracy and traceability of this standardization depend on many factors, and at small volumes (for which fluorometry
is most often used), standardization can be difficult. For this reason, fluorometry is most often used to determine precision
only, and not accuracy, leaving the user to estimate how close the actual dispense is to the desired volume. Work is currently
in progress to develop better traceability for fluorometric calibration methods.
Fluorescence methods are also affected by quenching and photo bleaching. Fluorescent dyes can chemically degrade over time
and are sensitive to temperature and pH. Some dyes are buffered, meaning they contain chemicals to prevent the pH from changing.
Unbuffered dyes, however, suffer from pH shifts as the dyes absorb carbon dioxide from the air and become acidic. This pH
shift can affect the accuracy of the measurement reading. Because the properties of fluorescent dyes can shift in the course
of hours, standard curves should only be relied on for short periods of time. In addition, no fluorometric calibration technologies
are commercially available, although some methods have been published in the scientific literature (3, 4).
In summary, fluorescent calibration is best suited for demonstrating precision in nearly identical conditions when testing
small liquid volumes and when accuracy and traceable measurements are not required.
Photometric calibration requires a photometer and stable dyes that absorb light in the visible or ultraviolet range. To use
single-dye absorbance photometry to measure volumes, a dye solution is delivered into a cuvette, a measuring cell, or a clear-bottomed
microtiter plate. A beam of light at a specified wavelength is passed through the solution, and the photometer measures the
quantity of light that passes through. The amount of light that is absorbed is proportional to the amount of dye present,
which permits a volume determination to be made.
The photometric method produces precise measurements and is less sensitive to environmental conditions than gravimetric and
fluorometric calibration technologies. In addition, although photometric dyes change with temperature and pH, they tend to
be more stable than fluorescent dyes. Hence, the response from the photometric reader will be comparatively consistent. In
addition, photometry is typically immune to other chemicals that can have a large impact on a fluorescence signal. Photometry,
therefore, is better suited than fluorometry for making accuracy determinations.
Another benefit of photometric calibration methods is the ability to provide information about each channel in a multichannel
device. Absorbance dyes that are readily available and commonly used include tartrazine and potassium dichromate. A commercially
available single-dye method for single-channel pipettes is commonly used in the clinical-laboratory industry.
ISO 8655-7 recognizes the technique of single-dye photometry for liquid-handling device calibration (5). According to this
standard, however, photometric methods should be accompanied by an uncertainty analysis that describes the measurement uncertainty.
This analysis may include error contributions such as accuracy of the photometer and reagents, dye instability, deviation
from ideal Beer's Law behavior, and the like.
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. Because light is passed through the sample and an optical
wall, the optical quality of the microtiter plate or cuvette used in the method can affect the accuracy and precision of the
measurement, and laboratories must also account for this.
Like all dye-based methods, photometric methods must be properly standardized to obtain quantitative results for accuracy
measurements. The traceability of the method depends on many factors, including how carefully the standardization is carried
out. For traceable photometric readings, a standard curve must be developed by using a known liquid-delivery device (e.g.,
a calibrated pipette) or by weighing volumes. This process can be time-consuming and tedious. In addition, it assumes that
the liquid-handling device used to develop the standard curve is reliable, and this assumption adds a level of uncertainty.
In summary, single-dye photometric calibration is well-suited for measuring precision, particularly when handling volumes
too small to be weighed on a balance. Accuracy measurements can also be made, but their robustness is limited because of the
difficulty of ensuring that the method is properly standardized and that an uncertainty analysis yields acceptable performance.