Laboratory scientists 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, that can be a very risky assumption with high costs of failure. Those 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 quality assurance is more critical than ever, laboratories require the most accurate, precise, and convenient methodology possible to verify the performance of their liquid handling instrumentation.

PRODUCT FOCUS: ALL BIOLOGICS

PROCESS FOCUS: R&D, ANALYTICAL

WHO SHOULD Read: QA/QC, R&D, AND PROCESS DEVELOPMENT

KEYWORDS: CALIBRATION, BIOASSAYS, LIQUID HANDLING, VALIDATION, CELL-LINE DEVELOPMENT, DRUG DISCOVERY

LEVEL: BASIC

In providing a method to measure light absorption and verify volume, ratiometric photometry has emerged as an answer. Highly accurate and precise even at low volumes, this technology provides laboratories with an easy-to-use process for quickly validating assay results and enhancing laboratory efficiencies. This translates into greater confidence in data, a cost reduction because repeated assays and remedial actions are eliminated, and more reliable regulatory compliance.

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 because its accuracy is affected by a variety of environmental factors including evaporation, static electricity, and vibration. The uncertainty grows as test volumes decrease. Fluorometry and single-dye absorbance photometry are other traditional methods of liquid delivery validation that also have technological limitations. As laboratories work with smaller and smaller volumes, the need for enhanced accuracy and precision grows.

Although the science behind ratiometric photometry might seem complex, this technology is easy to integrate and use day to day. Laboratories can now strengthen confidence in their assay results and enhance data integrity in minutes.

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The Science of Photometric Calibration

Photometry measures the absorbance of light by a dye solution at a given wavelength. To use it to verify small liquid volumes (e.g., 0.1 µL), the sample to be measured is combined with a larger volume of diluent solution. In this three-part process, the unknown sample volume is represented by the symbol V₁, the known diluent volume is represented by V₂, and the combined volume of both after mixing is represented by V₃. The diluent volume is known because it is measured using methods that are effective at larger volumes, such as through delivery by calibrated glassware.

The International Organization for Standardization has OFFICIALLY recognized the ratiometric approach to photometric calibration in a recently released standard.

It is important to note that in traditional single-dye photometry, dye molecules are present only in the sample solution, and there is no concentration of dye present in the diluent solution. To use photometry to verify liquid volume, the following conditions are applied: a mass balance equation, ideal mixing, and the Beer–Lambert Law.

Conservation of Mass (Mass Balance Equation): This fundamental law of chemistry can be applied to photometry provided that dye molecules in a liquid being measured are nonvolatile (Equation 1).

Conservation of Volume (or Ideal Mixing): This is a valid assumption as long as the chemical components of V₁ and V₂ are similar (Equation 2). For example, in most cases both are aqueous and contain small amounts of salts or organic molecules.

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), 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 a dye solution is measured and shown to be constant (Equation 3).

Those three 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 (Equation 4).

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

The accurate application of this formula to single-dye photometric calibration depends on the accuracy of absorbance measurements and knowledge of the diluent volume (V₂). So it’s difficult given that the absorbance standards used to calibrate photometers are limited in their accuracy.

Ratiometric Photometry

Ratiometric photometry off-sets the uncertainty of photometers by incorporating a second dye solution into the process, adding it to the diluent. Suppose that two dyes (e.g., one red and one blue) each have absorbance peaks at different analytical wavelengths. Place the red dye in the sample solution (V₁) and the blue dye in the diluent (V₂). Now the measuring 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 are known before mixing. After thorough mixing, the absorbance of red dye in the resulting mixture (A_R) is compared with the absorbance of blue dye in the original diluent (A_B) and expressed as a ratio. So this technology is thereby referred to as ratiometric photometry.

Building on the traditional single-dye photometric method described above, the second dye adds an additional mass balance equation and an additional Beer–Lambert equation. Algebraically combining those additional equations with the first three yields Equation 5.

Now each term inside parentheses is a ratio in itself.

A_R/A_B is the absorbance ratio measured by a 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. The volume of diluent solution (V₂) is determined in the same way as it would be in a traditional single-dye system. With all other quantities now known, the ratiometric photometry system can calculate V₁, the unknown volume delivered by the liquid delivery instrument being verified.

Benefits Over Traditional Methods

Ratiometric photometry offers several important advantages for reducing measurement uncertainty. First, the effects of optical imperfections are reduced in magnitude. Optical imperfections tend to influence absorbance readings in positive correlation. Thus, absorbance ratios are less influenced than individual absorbances by the most common sorts of optical imperfections.

Next, systematic inaccuracies in absorbance measurements have less effect on volume measurement with this photometry modification. Because both sample and diluent absorbances are measured using the same photometer, systematic errors in it tend to cancel each other out. Mathematically, systematic covariance in both the numerator and denominator of a ratio tend to offset one another, leading to relatively stable results.

Absorbance ratios can be measured more accurately than individual absorbances can, leading to a higher degree of accuracy and precision in ratiometric methods over traditional single-dye photometry. The underlying reason is that the absorbance of photometric calibration standards drifts over time, whereas ratios exhibit greater stability.

The second dye within the diluent in ratiometric photometry can be thought of as an internal standard that normalizes the method. This makes the technology 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, life-science laboratories now have an internationally approved methodology to verify the performance of their liquid handling instruments. The International Organization for Standardization (ISO) has specifically recognized the ratiometric approach to photometric calibration in a recently released standard (1). This development puts an international stamp of approval on an innovative technology, providing laboratories with a convenient quality assurance methodology that offers unparalleled accuracy and precision and will be applicable across the globe. The results are obvious: data integrity and greater efficiency.

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