The ratiometric photometric calibration method is a refinement of photometry designed to overcome the accuracy limitations
of traditional single-dye photometric volume measurements. Ratiometric photometry uses two standardized dyes, and its measurement
process produces absorbance readings in pairs that can be combined into absorbance-ratio readings.
The primary benefit of this approach is its ability to improve the accuracy and robustness of measurement comparison with
nonratiometric methods. Absorbance ratios can be measured more accurately than individual absorbances, leading to a higher
degree of accuracy and precision in ratiometric methods versus traditional single-dye photometric methods. The underlying
reason for the improved measurement ability of ratiometric photometric methods is that the absorbance of photometric calibration
standards drifts over time, while ratios exhibit greater stability.
Compared to gravimetry, this method offers greater speed, ease-of-use, and enhanced accuracy in small-volume measurements.
Compared to fluorometry, ratiometric photometry provides accuracy as well as precision measurements and can do so to a traceable
standard because the dyes function as an internal standard. Measuring the second dye in comparison to the first dye provides
a nearly automatic compensation for the most common photometric error sources.
Systems based on ratiometric photometry provide information about each individual channel in multichannel devices and good
plate-to-plate reproducibility. For ratiometric photometry to produce benefits, however, it must use well-characterized plates
and carefully calibrated solutions of good stability.
In addition, to function properly, ratiometric photometric methods require specially formulated dyes to produce accurate absorbance
ratios. Lastly, this technology is not always preferred when measuring only large volumes because other technologies may produce
adequate measurements more cost effectively.
In summary, ratiometric photometry calibrations provide great benefits when measuring small liquid volumes for protocols requiring
traceability and a high degree of accuracy per channel as well as precision.
Pharmaceutical laboratories have varying protocols, processes, and requirements, and these elements can affect the choice
of calibration technologies for liquid-handling devices. Gravimetry, fluorometry, single-dye photometry, and ratiometric photometry
are common means for verifying liquid-handling instrumentation. Each technique has its own advantages and disadvantages. Understanding
the assay and laboratory quality requirements, traceability needs, and tolerance for error as well as the level of accuracy
and precision required can help laboratories make the right decision.
Richard Curtis, PhD*, is chairman and chief technology officer, and George Rodrigues, PhD, is a senior scientific manager at ARTEL, 25 Bradley, Westbrook, ME 04092, tel. 207.854.0860, fax 207.854.0867, firstname.lastname@example.org
*To whom all correspondence should be addressed.
Submitted: Jan. 14, 2008. Accepted: Feb. 4, 2008.
1. ASTM International, "ASTM E1154-89 (2003) Standard Specification for Piston or Plunger Operated Volumetric Apparatus,"
(West Conshohocken, PA, 2003).
2. International Organization for Standardization, "ISO 8655-6: Piston-Operated Volumetric Apparatus—Part 6: Gravimetric
Methods for the Determination of Measurement Error" (Geneva, 2002).
3. P. Taylor et al., "A Standard Operating Procedure for Assessing Liquid Handler Performance in High-Throughput Screening,"
J. Biomol. Screen.
7 (6), 554–569, (2002).
4. J. Petersen and J. Nguyen, "Comparison of Absorbance and Fluorescence Methods for Determining Liquid Dispensing Precision,"
10 (2), 82–87, (2005).
5. ISO, "ISO 8655-7: Piston-Operated Volumetric Apparatus—Part 7: Non-Gravimetric Methods for the Assessment of Equipment
Performance" (Geneva, 2005).
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