Spectrofluorometers: Working Principles & Instrumentation
Spectrofluorometers are primarily used to take fluorescence measurements, or photoluminescence measurements. They are also capable of taking absorbance measurements.
Spectrofluorometers contain the same basic components as spectrophotometers: monochromators, a sample holder, a light source and a single pixel detector. However to measure fluorescence, they need an additional wavelength selection mechanism for the incident light, often a broadband light source combined with a monochromator. This stimulates the sample with a specific wavelength.
To perform florescence measurements, the first monochromator can be set to generate a fixed UV or blue light, while the second monochromator performs the wavelength scan over longer wavelengths. This allows you to measure the emission spectrum of your sample, probing information about its molecular structure.
How Does A Spectrofluorometer Work?
Spectrofluorometers have three main parts:
- An excitation source
- A sample holder
- A detector
Excitation Sources
For fluorescence spectroscopy, the excitation source must emit within a narrow wavelength range to selectively excite the fluorophore. Some spectrofluorometers use a narrowband light source, such as a UV excitation source. However, using a single wavelength source can limit the types of fluorophores you can measure, and can’t be used to measure excitation spectra.
Alternatively, some spectrofluorometers use a variable light source. This combines a broadband light source, such as a Xenon light source or a broadband LED, with a monochromator. The monochromator makes it possible to select specific wavelengths for sample excitation, allowing users to conduct a wider range of measurements. However, the strength of this method depends on the type of broadband light source used. Variable light sources may produce a less intense excitation signal, as only part of the power provided to a source is being used for the chosen wavelength.
Sample Holder
The sample holder is a straight-forward but vital part of the system. It ensures consistency between measurements and holds the sample steady. There are sample holders designed to measure substrates for thin film measurement. Additionally, many samples measure fluorescence within solution, usually in a quartz cuvette. Using ultra-flat quartz cuvettes or substrates helps to reduce reflection, scattering and absorbance of light. This ensures that the maximum possible light is transmitted through the holder without interaction.
Detectors
Spectrofluorometers use single pixel detectors, unlike spectrometers which use array detectors. Single pixel detectors can only measure intensity and have no way to decipher between different wavelengths of light. Therefore, a monochromator is positioned before the detector to select the wavelengths of light the detector is exposed to. Scanning monochromators are used to cycle through the different wavelengths of light, while the detector measures the intensity. This way the spectrofluorometer builds a fluorescence spectrum piece by piece.
Often photomultipliers are used as these detectors. The type of photomultipliers used will determine the spectral range and sensitivity of the spectrofluorometer.
What Measurements Can Spectrofluorometers Do?
Spectrofluorometers can perform a variety of measurements:
- If the excitation wavelength is fixed and the emission wavelength is varied, spectrofluorometers can measure the emission spectrum of a fluorophore.
- If the emission wavelength remains the same but the excitation wavelength is varied, spectrofluorometers can measure an excitation spectrum.
- If these two monochromators are synchronized i.e. set to the same wavelength during a measurement, the system can measure absorbance, much like a spectrophotometer – but with one advantage. Since the fluorescence emission is at a longer wavelength than excitation, this second monochromator will filter out any fluorescence generated by light from the first monochromator.
Fluorescence Spectroscopy
One of the most common uses of spectrofluorometers is for fluorescence spectroscopy. Fluorescence spectroscopy is a vital technique used in many fields of research. Most prominently, it is used to study the behavior of fluorophores, either for use in fluorescent applications, such as in LEDs, or as a marker to study biological processes or chemical reactions.
Fluorescence spectroscopy analyzes the light emitted from a sample as an excited electron relaxes from the first electronic level into the ground state. There are two types of fluorescence measurement: steady state fluorescence and time resolved fluorescence. Time resolved PL measurements are used to measure dynamic processes like fluorescence lifetime. Steady state fluorescence can be used to probe the molecular structure of a material and is measured using a spectrofluorometer.
Fluorescence spectroscopy is often used alongside absorbance spectroscopy, as absorbance measures the excited state of a molecule whereas fluorescence measurements probe the ground state.
Spectrofluorometer vs. Spectrophotometer
Spectrofluorometers are similar to spectrophotometers - often the terms are used interchangeably. They use the same components, and they build a spectrum in the same way.
The main difference between spectrofluorometer and spectrophotometers is that spectrofluorometers are capable of measuring fluorescence or photoluminescence, as well as absorbance. For fluorescence spectroscopy, a spectrofluorometer needs the ability to select the incident radiation wavelength, which requires both excitation and emission wavelength selection. A spectrophotometer has only 1 monochromator and uses a broadband light source, so can only be used for absorbance measurements.
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How Does A Spectrophotometer Work?
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