Ratiometric Photometry Improves Laboratory Quality Assurance

New Methodology Accurately and Precisely Verifies Low Volume Liquid Delivery

Laboratories often assume that their liquid handling instruments, from pipettes to automated liquid handlers, are operating within specification. But given that data integrity for applications from drug discovery to molecular diagnostics relies on accurate and precise liquid delivery, this is a very risky assumption with high costs of failure. These costs and risks are compounded by several trends in today’s life science laboratories, such as the growing use of valuable reagents at low volumes and an increasingly strict regulatory environment. Because the need for liquid delivery quality assurance is more critical than ever, laboratories require a more accurate, precise and convenient methodology to verify the performance of liquid handling instrumentation.

Ratiometric Photometry has emerged as the answer, measuring light absorption to verify volume. Highly accurate and precise, even at low volumes, this technology provides laboratories with an easy-to-use process to quickly validate assay results and enhance laboratory efficiency. This translates into greater confidence in data, a reduction in costs due to elimination of repeated assays and remedial action, and more reliable regulatory compliance.

As an alternative to Ratiometric Photometry, liquid delivery instrumentation can be verified using gravimetry, which measures liquid weight on analytical balances. Requiring lengthy and tedious calculations, this manual method is ineffective at verifying low volumes. This is because the accuracy of gravimetric processes is impacted by a variety of environmental factors, including evaporation, static electricity and vibration. And this uncertainty grows as test volumes decrease. Fluorometry and single-dye absorbance photometry are other traditional methods that also have technological limitations. As laboratories work with smaller and smaller volumes, the need for enhanced accuracy and precision grows.

This article describes the basic science underpinning traditional photometric volume measurements as well as how Ratiometric Photometry builds on this foundation to overcome accuracy and precision problems encountered with traditional methods of volume measurement. While the science behind Ratiometric Photometry might seem complex, this technology is easy to integrate and use on a day-to-day basis. As a result, laboratories can now strengthen confidence in their assay results and enhance data integrity in minutes.

The Science of Photometric Calibration

Photometry measures the absorbance of light by a dye solution at a given wavelength. To use photometry to verify small liquid volumes, such as 0.1 microliters, the sample to be measured is combined with a larger volume of diluent solution. This is a three-part process where the unknown sample volume is represented by the symbol V1, the known diluent volume is represented by V2 and the combined volume of sample and diluent after mixing is represented by V3. The diluent volume is known because it is measured using other methods that are effective at larger volumes, for example through delivery via calibrated glassware.

It is important to note that in traditional single-dye photometry, dye molecules are present only in the sample solution. Thus, there is no concentration of dye present in the diluent solution.

To use photometry to verify liquid volume, the following conditions are applied:

  • Conservation of Mass (Mass Balance Equation): This fundamental law of chemistry can be applied to photometry provided that the dye molecules in the liquid being measured are non-volatile.

Figure 1:   V1C1 + V2C2 = V3C3

  • Conservation of Volume (also called Ideal Mixing): This is a valid assumption as long as the chemical components of V1 and V2 are similar (e.g., both are aqueous-based and contain small amounts of salts or organics).

Figure 2:  V1 + V2 = V3

  • Absorbance is Proportional to Concentration (Beer-Lambert Law): Simply stated, the Beer-Lambert Law says that when light is passed through a solution containing some concentration of dye, the amount of light absorbed by the dye solution is proportional to both the molar absorptivity (ε λ), which is a physical property describing molecular light absorbance, and the concentration of the dye (C), as well as the path length of light (l) through the solution. To apply this law, the molar absorptivity of the dye solution needs to be measured and shown to be constant.

Figure 3:  Aλ = ΕλlC

The three above equations can be algebraically combined to yield the following equation, which constitutes the simplest form of volume calculation applicable to traditional single-dye photometric volume measurement:

Figure 4: V1 = V2 [A3/(A1-A3)]

Thus, to measure the sample volume (V1), a photometer is used to measure A1, which is the absorbance of the undiluted sample solution, and A3, the absorbance of the final mixed volume. The volume of the diluent (V2) also must be known by some means such as delivery from calibrated glassware.  The sample volume dispensed by the liquid handling instrument in question, V1, can then be calculated.

The accurate application of this formula to single-dye photometric calibration is contingent upon the accuracy of the measurement of the absorbances, and knowledge of the diluent volume (V2). This is difficult given that the absorbance standards used to calibrate photometers are limited in accuracy.

Ratiometric Photometry

Ratiometric Photometry offsets the uncertainty caused by the photometer by incorporating a second dye solution into the process, adding this second dye to the diluent. As an example, suppose that two dyes (e.g., one red, one blue) each have absorbance peaks at different analytical wavelengths. For the purpose of explanation, we will place the red dye in the sample solution (V1) and the blue dye in the diluent solution (V2). The technique is straightforward: an unknown volume of the red dye solution is delivered into a known volume of blue diluent.  The concentration, hence absorbance, of both blue and red dyes prior to mixing are known.  After thorough mixing, the absorbance of the red dye in the resulting mixture (AR) is compared as a ratio to the absorbance of the blue dye in the original diluent (AB). The technology is thereby referred to as Ratiometric Photometry.

Building on the traditional single-dye photometric method, the second dye adds an additional mass balance equation and an additional Beer-Lambert equation. Algebraically combining these two additional equations with the three equations previously presented yields the below equation:

Figure 5: V1 = V2 [(AR/ AB)/(K-(AR-AB))]

Now, each term inside the parentheses is itself a ratio. AR/AB is the absorbance ratio measured by the Ratiometric Photometry system. K is the pure absorbance ratio of both dye solutions before mixing, combining concentration information for both the red and blue dyes. V2, the volume of diluent solution, is determined in the same way as in a traditional single-dye system.

With all other quantities now known, the ratiometric photometry system is able to calculate V1, the unknown volume delivered by the liquid delivery instrument being verified.

Benefits of Ratiometric Photometry versus Traditional Methods

The Ratiometric Photometry method offers several important advantages for reducing measurement uncertainty:

  • The effects of optical imperfections are reduced in magnitude with Ratiometric Photometry.  This occurs because optical imperfections tend to influence absorbance readings in a positively correlated way.  Thus, absorbance ratios are influenced less than individual absorbances by the most common sorts of optical imperfections.
  • Systematic inaccuracies in the absorbance measurements have less impact on the volume measurement.  Because both sample and diluent absorbances are measured using the same photometer, systematic errors in the photometer tend to cancel out.  Mathematically, systematic covariance in both the numerator and denominator of a ratio tend to offset one another, resulting in a more stable result.
  • 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 this is that the absorbance of photometric calibration standards drifts over time, while ratios exhibit greater stability.
  • The second dye within the diluent can be thought of as an internal standard that normalizes the method. This internal standard makes the ratiometric method more robust and practical in application.  Consequently, Ratiometric Photometry lends itself to reliable application as a standard method across different locations and laboratories.

International Approval

Using ratiometric photometry, laboratories now have an internationally approved methodology to verify the performance of liquid handling instrumentation. This is because the International Organization for Standardization (ISO) has specifically recognized the ratiometric approach to photometric calibration in its recently released standard ISO 8655-7. This development puts an international stamp of approval on this innovative technology, providing laboratories with a convenient laboratory quality assurance methodology with unparalleled accuracy and precision that is applicable across the globe. The result: data integrity and greater efficiency.