Ultra-compact Device is Ideal for Measuring Color-inducing Reagents
Spark is a very small spectral sensor from Ocean Optics that bridges the spectral measurement gap between filter-based devices such as RGB color sensors and CCD-array instruments such as miniature spectrometers. As this application note explores, Spark provides a simple tool for measuring color-based analytes such as pH indicators and other chemical reagents.
Spark is a visible spectral sensor (380-700 nm) that operates as a stand-alone device or as part of an embedded system. This makes it attractive for lab use and as part of handheld instruments and in-line testing systems. Absorbance, fluorescence and color measurements are possible.
As a visible spectral sensor, Spark is well suited to measurement of colored samples (both shade and intensity) and, as such, is useful for color-matching applications. Unlike a traditional color sensor, which provides an estimated response based on output from individual detectors linked to a primary (often RGB) color filter, Spark records the actual spectral response of a colored sample (Figure 1) and captures significantly more information.
At the heart of the Spark is a proprietary solid state component that is packaged within a protective enclosure (Fig 2A) or available as a basic electronic component (Fig 2B). In the latter format it is easy to appreciate how such a device can be integrated into a measurement system such as a portable tester, or built into a sample flow or dispensing system, especially where reagents are used. Indeed, spectral sensing is a powerful tool for measuring reagent reactions, as samples can be measured in situ and with minimal reagent consumption.
There are many examples of measurements made possible by the use of color inducing reagents: changes in pH levels, interaction with certain ions, chemical complex formation (such as with certain metals), and oxidation or reduction reactions. Such measurements are widely used in diagnostic and chemical or biological test kits, often in the form of a test strip or a dipping stick. Other uses of specific reagents may be in the form of molecular, chemical or biological taggants – i.e., materials added to give specific responses based on color changes or the generation of fluorescence when viewed with an excitation light source.
The Importance of Color and Reagent-based Measurements
Color measurements following some form of chemical interaction were some of the first published analytical methods developed based on spectral measurements. Originally, such methods were classified as colorimetric or photometric and typically linked to a filter-based measurement. One of the earliest devices for such measurements (in the 1890s) was the Lovibond Tintometer, where an assessment of sample coloration was evaluated based on a standardized color response. The Tintometer output, which is still used as a commercial standard for evaluating the “color” of a liquid sample (or transparent solid), is expressed as a color index or a standardized value. This value is based on a proprietary procedure based on a visual assessment of a sample, when viewed in “standard” light, against one of 84 glass standards. These standards are in place today and are used for color assessment of food and beverage products, as well as materials such as plastics, oil and petroleum products.
Color assessment is still a critical parameter for many products, especially in consumer industries, where an off-color or color cast is readily detected. Also, some materials discolor with time and as a result of oxidation, with meats and produce, for example, evaluated for freshness on the basis of color changes. The challenge is how to detect and quantify these characteristics. Implementing a simple spectrometer-based tester for product and quality testing purposes would be beneficial throughout production processes, from incoming materials inspection to final distributed products.
Color analysis also plays a role in medical diagnostics and life sciences applications. For example, yellow pigmentation of the skin can be associated with liver disorders, including jaundice (bilirubin) in newborn children. Urine testing is used as a medical diagnostic based on color tests for pregnancy and assessment of leukocytes, nitrite, urobilinogen, protein, pH, hemoglobin, ketone and glucose.
Handheld instruments are available for the measurement of sample colors on surfaces (by reflection) or through bulk samples (by transmission). Some systems use a tristimulus approach based on RGB (red-green-blue) responses; others are extensions of light meters with associated color filters. The latter provides only an approximation of color values, whether the color density is being measured or the color produced by a reagent reaction is being measured.
Examples of Color-based Reagent Applications
Invoking a color change by the addition of a reagent is a common mode of measurement. The simplest reagent in this context is the use of a color indicator for pH measurements. In analytical terms, a color indicator is used in acid-base neutralization, such as with methyl red (Figure 3) and phenolphthalein (Figure 4). The transition for methyl red occurs at a pH of 5.0, and that for phenolphthalein at a pH of 9.6. In the case of methyl red the color transition moves from red to yellow with orange as the intermediate color of the mixed forms (acid-base). We observe a red color (centered at ~523 nm) for acidic solutions, and a yellow color (~445 nm) for neutralized solutions (pH >5.0); the yellow curve in Figure 3 shows the transition between the two forms with the mixed forms being present.
Phenolphthalein is a unique indicator because the acid form is colorless, and the transition to the base form occurs with the appearance of the peak at 545 nm (pink coloration), at a relatively high pH (9.6). Monitoring the acid-base neutralizations spectrally can be accomplished in real-time and reproducibly, adding insight and complementing potentiometric titration methods.
Color indicators are commonly used for pH measurements, where the color transitions are used to visually estimate the pH of a water sample such as swimming pool water. Figure 5 illustrates this application where the transitions from pH 6.0 to pH 7.6 are shown with a series of overlapped spectral curves, with the transition from yellow to blue with shades of green as intermediate pH values.
Measurements of color transitions are not limited to pH changes. For example, when combined with a chelating agent, reagent color transitions can be used to measure dissolved metals and oxidizing agents. For these applications, Spark can be used as a lab instrument or as a component in a microfluidics or similar system, providing the benefits of miniaturization, disposable sampling, minimal loss of reagents and less waste.
In the past, simple colorimetric reagent reactions have been monitored by basic filter photometers, where an average absorption or extinction is measured. This is the basis of many low-cost test kits used today for laboratory and field based testing. These kits are limited to a single analyte, measured one at a time, or to a range of analytes where non-specific properties or materials such as oxidants or heavy metals are measured.
With the versatile Spark, multiple, simultaneous measurements of multiple analytes are possible. In this case, multiple analytes are measured as a mixture; the measured spectrum contains overlapping absorptions. Also, multiple reagents may be used in a system enabling multiple analyses to be performed in situ and in parallel.
The applications mentioned to this point can be defined as chemical based reactions, where the chemistry can be optimized and reduced in size and volume. The ability to mix reagents and/or handle multiple analytes places the Spark-based detection system in a unique position relative to mobile sensing and testing, or integrated (embedded) testing. Beyond acid-base and metals detection, other chemical agents and chemical reactions that work well are oxidants or oxidizing agents. Good examples are the measurement of chlorine content, as used to measure residual levels of chlorine in treated municipal water (or even swimming pool water), and determination of dissolved oxygen content. The latter can be important in measuring the aeration of water, and the health of lakes and streams. Examples of both measurements are provided in Figures 6 and 7. In both cases concentrations as low as fractional ppm can be measured.
Color reactions can result from molecular associations and interactions, as are common in medical and clinical applications. As described earlier, jaundice is indicated by the characteristic yellowing of the skin (and the whites of the eyes) and is caused by the presence of bilirubin, an orange-yellow pigment formed by the breakdown of hemoglobin in the liver. Bilirubin has a characteristic spectral response with absorption centered between 450-460 nm (Figure 8). The spectrum shown is for a calibration standard for a working range up to bilirubin values of ~20 (mg/dL). The bilirubin may be measured in blood samples, in body waste products and noninvasively through the skin. With the full spectral response of Spark, bilirubin can be distinguished from potential interferences such as hemoglobin and blood-based derivatives.
In addition to reagent-based chemistry focused on color reactions, Spark can be used to measure reagents that combine with a reactive functional group to produce a modified chemical species, where the newly formed species is fluorescent. This approach is the basis of taggants that are used to measure and detect specific chemical and biological reactivity, and is often used for differentiation of proteins and protein-based materials, including bacterial materials.
For such applications, the reagent is added and the formation of fluorescence can be monitored with the Spark detector, when the sample is viewed under UV light (typically 365 nm). Also, the interaction with the reagent may be monitored by fluorescence quenching. In this case a diminution of the level of fluorescence is monitored.
Summary: Chemical Reagents and Color Measurements
Various applications related to chemical reagents have been cited in this application note, ranging from pH measurements to the detection and measurement of biologically important materials. Here are some other examples:
- Water analysis – from surface waters to wastewater and industrial discharge
- Food and beverage analysis – from contaminants and spoilage byproducts to additives and allergens
- Soil analysis – for contamination and remediation qualification measurements
- Medical and clinical – for medical diagnostics and clinical testing of bodily fluids
- Chemical vapor – for vapor-chromic detection, where reactive gases or vapors are immobilized on an optical element or a membrane
There are many other relevant applications, with the compact size of Spark adding flexibility to the measurement system form factor. Ultimately, thanks to its novel design elements and full spectral response, Spark offers a simple yet elegant tool for chemistry-based color and other measurements.