Water bands due to minute amounts of water vapor in the glass cause absorption, and scattering occurs when light bounces off molecules within the glass. To reduce light absorption, the refractive index of the fiber optic core must be greater than the refractive index of the cladding.
Fiber optic cables are most often applied to NIR and IR studies, which frequently have sources that cannot be transferred to a cuvette. The most common fiber optic wavelengths are nm, nm, and nm. The spectral resolution of a spectrometer refers to its ability to resolve spectral features and bands into their respective components. In dispersive array spectrometers, spectral resolution is dependent on the slit, diffraction grating, and detector.
The slit determines the minimum image size that the optical bench can form on the photodiode. The diffraction grating determines the total wavelength range of the spectrometer, and the detector determines the maximum number and size of discrete points that can be digitized. If the spectral resolution is too low for an experiment, then the data will be missing key points.
A high resolution spectrometer can extend the total measurement time, but the quality of the data is optimized. While there are many types of spectrometers, all spectrometers take in light, split it into its spectral components, digitize the signal as a function of wavelength, and display it through a computer.
The design of a spectrometer changes depending on the scope and intentions of the experiment, allowing researchers to measure molecular vibrations, absorbance, mass-to-charge ratios, and much more. A monochromator is structurally similar to a spectrometer, but provides a much smaller window of data.
A monochromator captures one measurement in the UV-VIS spectrum at a particular, predetermined, wavelength or bandwidth. Alternatively, a spectrometer captures the entire UV-VIS spectrum in the same amount of time, and provides values for each wavelength.
Radiometers consist of a meter body that measures current voltage from an internal or external detector. A sensor or photodiode is used to measure a specific band of light, and filters are added to the sensor to block unwanted wavelengths. Radiometer sensors are calibrated at the desired peak intensity and measure all of the light under the curve to generate a single reading. A spectrograph is an instrument that separates light by its wavelength or frequency and records the resulting spectral range in a multichannel detector, such as a photographic plate.
Light entering a spectrograph through a small opening in the spectrograph hits a collimating mirror that lines up the entering rays of light parallel to each other.
Then, the rays hit a diffraction grating, passing through or bouncing off into their constituent wavelengths, each with their own speed and direction that are dependent on their spectral color.
The grating bends each wavelength in a different direction, separating red from orange, orange from yellow, and so on. The diffraction grating controls can be rotated to change which wavelengths of light reach a second mirror, which then focuses them onto a photodetector that converts photons into electrical signals for computer analysis.
Spectroradiometers are ideal for measuring the spectral energy distribution of small, precise light sources. Light is dispersed using prisms or diffraction gratings. Spectroradiometers record the radiation spectrum of a light source and calculate parameters such as luminance and chromaticity. Factors such as sensitivity, linearity, stray light, and polarisation error are less influential on spectroradiometry than spectrometry, making spectroradiometry a more efficient method for measuring narrow-band emitters.
A spectroscope is a hand-held device used to identify the spectral composition of light. Early astronomers used spectroscopes to study the composition of planets and stars. The spectrums observed by these astronomers played a key role in dozens of hypotheses about the gaseous nature of planets within our solar system. Spectrophotometry measures how much light is absorbed by, reflected off, or transmitted through a chemical substance by measuring the intensity of light as the beam passes through a sample.
Electromagnetic energy from the sample, enters the device through the aperture and is separated into its component wavelengths by holographic grating. The separated light rays are focused onto a CCD array detector which determines the intensity of each wavelength using a pixel of the array. Spectrophotometry has broad applications within science and is used within biochemistry, physics, material and chemical engineering, clinical application, and chemistry.
Spectrophotometers can be divided into two categories that are dependent on the wavelength of the light source. UV-Visible spectrophotometers use wavelengths of light that are higher than the ultraviolet range - nm and visible range - nm of the electromagnetic spectrum. This type of absorption spectroscopy targets the transition of molecules from the ground state to the excited state.
UV-VIS spectroscopy is commonly used by analytical chemists for the quantitative determination of different analytes, such as organic compounds, macromolecules, and metal ions. IR spectrophotometers use light wavelengths in the infrared range - nm of the electromagnetic spectrum.
Mass spectrometry can be used to identify molecules within a sample, detect impurities, analyze a purified protein, or study the protein content of cells. Mass spectrometers use these three components for their measurements: ionization source, mass analyzer, and ion detection system. The ionization source converts molecules to gas-phase ions via vaporization before manipulating them with external electric and magnetic fields. The mass analyzer sorts and separates ions according to their mass-to-charge ratios using acceleration and deflection.
Each band correlates to vibration frequencies that are related to a change in dipole moment between the bonds of the atoms within the sample. NIR spectroscopy usually requires a high resolution spectrometer to ensure accurate data.
Each chemical element reacts slightly differently in this process, some visibly those on the mm wavelength that are detectable to the human eye and some invisibly like infrared or ultraviolet waves, which are outside the visible spectrum. As each atom corresponds to and can be represented by an individual spectra, we can use the analysis of wavelengths in the light spectrum to identify them, quantify physical properties, and analyse chemical chains and reactions from within their framework.
Spectroscopy is the science of studying the interaction between matter and radiated energy. On the other hand, spectrometry is the method used to acquire a quantitative measurement of the spectrum.
In short, spectroscopy is the theoretical science , and spectrometry is the practical measurement in the balancing of matter in atomic and molecular levels.
This could be a mass-to-charge ratio spectrum in a mass spectrometer, the variation of nuclear resonant frequencies in a nuclear magnetic resonance NMR spectrometer, or the change in the absorption and emission of light with wavelength in an optical spectrometer.
The mass spectrometer, NMR spectrometer and the optical spectrometer are the three most common types of spectrometers found in research labs around the world. A spectrometer measures the wavelength and frequency of light, and allows us to identify and analyse the atoms in a sample we place within it. In their simplest form, spectrometers act like a sophisticated form of diffraction, somewhat akin to the play of light that occurs when white light hits the tiny pits of a DVD or other compact disk.
Light is passed from a source which has been made incandescent through heating to a diffraction grating much like an artificial Fraunhofer line and onto a mirror. As the light emitted by the original source is characteristic of its atomic composure, diffracting and mirroring first disperses, then reflects, the wavelength into a format that we can detect and quantify.
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Figure 1: The three most common types of spectrometers found in research labs around the world. The goal of any optical spectrometer is to measure the interaction absorption, reflection, scattering of electromagnetic radiation with a sample or the emission fluorescence, phosphorescence, electroluminescence of electromagnetic radiation from a sample.
Optical spectrometers are concerned with electromagnetic radiation that falls within the optical region of the electromagnetic spectrum which is light spanning the ultraviolet, visible and infrared wavelength regions of the spectrum. In order to gain maximum information, the interaction or emission of light should be measured as a function of wavelength and the common feature of all optical spectrometers is therefore a mechanism for wavelength selection.
In low cost spectrometers or in situations where accurate wavelength selection is not important, optical filters are used to isolate the wavelength region of interest. However, for accurate wavelength selection and the generation of spectra, a dispersive element that separates light into its constituent wavelengths is required.
In all modern spectrometers, this dispersive element is a diffraction grating where constructive and destructive interference is used to spatially separate polychromatic light that is incident on the grating Figure 2. Figure 2: Dispersion of light into its constituent wavelengths by a diffraction grating.
Diffraction gratings are a key component of a monochromator, which is a device used to select a particular wavelength of light from a polychromatic light source. In a monochromator the diffraction grating is rotated to change the wavelength that aligns with and passes through the exit slit.
Excitation monochromators are found in all spectrophotometers see following section for selecting the desired excitation wavelength to reach the sample from a white light source Figure 3 Excitation Monochromator and spectra are measured by scanning the monochromator and measuring the change in signal as a function of excitation wavelength.
For detecting the light emitted by a sample there are two approaches. The first is an emission monochromator which works using the same principle as above except the light source is the emission from a sample and the monochromator selects which wavelength of light reaches the detector Figure 3 Emission Monochromator.
At least one emission monochromator or spectrograph is found in all spectrofluorometers and Raman spectrometers see following sections. Figure 3: The basic operating principle behind monochromators and spectrographs. It should be noted that these images are highly simplified for illustration.
For example the monochromators used in the Edinburgh Instruments FS5 and FLS are a more complicated Czerny-Turner design which have two slits and two ellipsoidal mirrors for superior performance but the principle remains the same.
Now that the key component of a spectrometer has been identified, the different types of spectrometer, their role, and basic design can be discussed. Three of the most common optical spectrometers: spectrophotometers, spectrofluorometers and Raman spectrometers are introduced. The term spectrophotometer can refer to quite a variety of instruments that measure light, with the exact definition depending on the area of science or industry.
Within academic research particularly chemistry and biology laboratories the term spectrophotometer is used specifically to refer to a spectrometer which measures the absorption of light by a sample and that definition will be used here. Figure 4: Simplified diagram of a single beam spectrophotometer. A stylised layout of a basic single beam spectrophotometer is shown in Figure 4.
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