In this article, we’ll uncover a few ways to analyze the visual and molecular characteristics of a sample. Color measurement technology comprises a multitude of closely related instruments and photometric techniques that analyze the properties of a sample in terms of color, brightness, and other visual characteristics. Fluorescent-based techniques provide a wealth of information about a sample's atomic and molecular characteristics. I’ll discuss color measurement first and then describe the fluorescent technologies.
The most common application for color measurement instruments is the analysis of solid and printed materials. However, other suitable devices may also be used to analyze liquid, slurries, powders, and more. It is important to note that filter photometers measure concentration in liquid samples contained in cuvettes while colorimeters are specifically used for the analysis of the color of the sample itself. Such measurements are based upon a predetermined color standard that has been developed to model color as perceived by the human eye.
Color spectrophotometers use photometers to measure the intensity of the transmittance and/or reflectance of a light beam as a function of its wavelength. To achieve this, most devices will fracture the visible spectrum into discrete bands and compare the transmittance and/or reflectance of each band as it interacts with the sample and a standard. The intensity of the distinct beams from the sample and standard are then analyzed and used to generate linear transmission and/or absorbance/concentration values. Spectroradiometers, which do not include an integrated light source, are another variation on the spectrophotometer. Colorimeters use fixed filters to collect the intensity of the three-color spaces.
Densitometers detect the strength of transmitted light, thus measuring the total optical density of a sample. These devices are commonly used for examining films, such as medical x-rays. Densitometers are distinct from devices that directly measure mass density of a sample. Color measurement devices include specialized tools that can analyze gloss, texture, and other surface qualities of a sample.
While color measurement technologies analyze the visible features of a sample, fluorescence and luminescence technologies provide information about the atomic and molecular characteristics of a sample and its interactions. Unlike absorption-based techniques such as UV and visible spectrometry which use only one wavelength, fluorescent-based techniques use two separate wavelengths. One is the excitation wavelength, the other, the emission wavelength. The energy of a sample is first elevated using a light source with a particular excitation wavelength, and then partial emission of that energy from the sample, the fluorescence, is measured at a specific emission wavelength.
Since this technology uses two wavelengths, it is extremely specific. In fact, the exact combinations of complex molecules with only slight differences in their molecular structures can be detected by fluorescence spectroscopy. Also, due to the isotopic nature of the fluorescent phenomenon, the emitted light can be captured orthogonally to the excitation light, resulting in a more dynamic range for detectors, and augmented sensitivity.
Fluorometers can be classified into three types: spectrofluorometers, filter fluorometers, and fluorescent lifetime. Spectrofluorometers are built using monochromators and, thus, are capable of measuring and scanning a range of wavelengths. On the other hand, filter fluorometers use individual fixed filters, covering only narrow wavelength bands. While spectrofluorometers are designed for speed and general use, filter fluorometers are designed for precision and analysis of specific compounds. Lifetime fluorescence systems comprise another category of fluorometers. Rather than measuring the wavelength and intensity of the fluorescing energy, these instruments measure the average time a molecule stays in an excited state before emitting a photon and returning to its ground state.
I will also mention luminescence, which involves the measurement of chemiluminescence and bioluminescence. Chemiluminescence is the light that is emitted from a chemical reaction, while bioluminescence stems from biological reactions, usually enzyme-catalyzed activity.
Color measurement, fluorescence, and luminescence can be used in the identification and classification of a sample. It is ultimately up to the user as to which technology will work best for his or her field of research.
Have you used any of these techniques in your research? What did you perceive to be a pro or con of the technology? Start the discussion below!
Disclosure: LabPulse.com is a sister company of the Science Advisory Board.