Electrostatic effects also cause uncertainty with gravimetric methods because plastic pipette tips are typically used to transfer
liquids. The static electricity that is imparted to the balance pan or the draft shield induces a force that affects measurement
accuracy. When working with small volumes, the error can be significant. Vibration must also be controlled for, often by calibrating
the instrument in a controlled environment on a solid marble bench.
Because gravimetric measurements calculate volume by converting weight to mass and then to volume, accurate calibration is
contingent on knowing the density of the fluid being pipetted. Many laboratory technicians assume the fluid being measured
has the density of 1 g/mL, which is the approximate density of water. Although common solutions do have published density
values, the densities are not always known to a high degree of accuracy.
To illustrate the possible uncertainty, consider dimethyl sulfoxide (DMSO). Its published density is 1.1 g/mL. The density
is published with limited resolution, using only two significant figures. In addition, the density of DMSO changes according
to its water content, which depends on the starting water content and the time it is exposed to ambient local temperature
and relative humidity. Even the density of water varies with temperature and, at room temperature, is always less than 1 g/mL,
its commonly accepted value.
These details must be accounted for if precise measurements are required. Consider a device with accuracy specifications of
better than 0.6%, which is a typical specification for high-accuracy pipetting of 1000 μL. Failure to correct for density
errors, even when pipetting water, can lead to error in the 0.3–0.5% range, which is nearly as large as the acceptable error
for the entire piece of equipment. When acceptable tolerances are in the 5–10% range, however, density considerations are
much less important.
An alternative to referring to published density values is to measure density with a commercial densitometer or pycnometer.
For reliable results, these instruments require calibration as other laboratory instruments do. Care must be taken to avoid
measurement error.
One last drawback of gravimetry is its inability to simultaneously measure each individual channel in multichannel liquid-handling
devices. With gravimetry, individual aliquots can be measured or multiple dispenses may be made and the total weight used
to calculate the average volume. To measure the performance of single channels, each channel must be tested one tip at a time.
This process is time-consuming and tedious. Testing each channel one time in a 96-channel device, for example, would require
96 dispenses.
In summary, gravimetric calibration is best suited to measuring the performance of single-channel devices that handle large
liquid volumes, usually those above 200–1000 μL. The precise lower limit for effective use of gravimetry depends on the tightness
of the tolerance to be met and the quality of the measuring equipment and procedure used.
Fluorometry
During fluorometric calibration, a beam of ultraviolet light is shone on a sample at one wavelength, called the excitation
wavelength. This exposure causes the molecules to absorb light and enter an excited electronic state. Release of this excess
energy results in the emission of light at a different, longer wavelength, called the emission wavelength. A detector is used
to measure how much light is emitted at the emission wavelength. Precision is measured by comparing relative fluorescence
levels in different samples.
Fluorescent dyes are photoactive and generate a strong signal at low volumes. Small samples can generate large signals at
low concentrations, which facilitates fluorometry's use in measuring small volumes. Measurements of volumes as small as 5
nL are possible.
A major drawback of fluorometry is the difficulty in achieving robust traceability, which often prevents its use in regulated
laboratories. This deficiency results from the fact that the strength of the fluorescent measuring signal varies depending
on the local chemical environment. Factors such as solvent composition, pH, ionic strength, redox potential, and time can
alter the signal strength. For this reason, during a given measurement, the volume in a well can be compared with a volume
in the previous well, provided that all volumes have similar chemical compositions. It is difficult, however, to compare measurement
readings day-to-day, assay-to-assay, or location-to-location unless traceability is established. Traceability is typically
established by developing a standard response curve using a calibrated pipette or other traceable liquid-delivery device.
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