How Does A Spectrophotometer Work?
A spectrophotometer is a piece of equipment used to quantify the absorbance of light by a sample. Spectrophotometers can be used to:
- Identify materials by mapping molecular absorption profiles.
- Work out solute concentrations of solutions.
- Detect trace impurities in samples or follow the progress of chemical reactions.
This makes spectrophotometers an invaluable resource in many industries ranging from food and beverage, cosmetics, environmental monitoring, biotechnology, chemical engineering and physics.
There are four important components within a spectrophotometer: a light source, a sample chamber, a monochromator and a detector. Light from a broadband light source is focused onto the sample that sits within the sample chamber. The transmitted light enters a monochromator, before being detected by the photodetector. The monochromator uses a rotating diffraction grating to adjust the wavelength of the transmitted light hitting the detector. This allows the spectrophotometer to build an absorbance spectrum.
Components of a Spectrophotometer
The four essential components to a spectrophotometer are as follows:
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A Light Source
This is generally a broadband (white) light source e.g. halogen bulb, arc lamp or white LED, as this allows the absorption of a sample to be measured at multiple wavelengths and an absorption spectrum to be recorded. It is possible to use a monochromatic light source (i.e. a laser), but in this case you can only measure absorption at a specific wavelength.
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A Sample Chamber
The sample sits within the sample chamber, which may be designed to hold a specific type of sample e.g. a thin film or an optical cuvette or may have interchangeable sample holders for greater flexibility. It is important that the sample is placed in the same position and orientation every time to keep consistency between measurements. The sample holder and surrounding optics provide this consistency.
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A Monochromator
The light from the sample needs to split into its component wavelengths so that intensity can be precisely measured by the detector. This splitting is performed by a monochromator, which consists of an entrance slit, a diffraction grating (or less commonly a prism). The diffraction grating can rotate, directing the a different portion of the optical spectrum towards an exit slit. This allows you to select the output wavelength.
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A light detector
Once light has passed through the sample, its intensity needs to be quantified, using a photodetector. In short, a photodetector will convert incident light into an electrical signal. This electrical output will be proportional to the incident light input. There are several types of photodetectors, suitable for different wavelengths and/or intensities of light. Silicon-based detectors are a common choice for visible light measurements. Additional electronics are also required to turn the signal from the photodetector into a meaningful number.
Spectrophotometer Theory of Operation
Spectrophotometers operate on the principles of absorbance spectroscopy, a technique grounded in the Beer-Lambert Law. This law relates the amount of light absorbed by a sample to the properties of that sample and the path length of the light. Specifically, absorbance (A) is given by:
Where:
- ε is the molar absorptivity (a constant for a given substance and wavelength)
- c is the concentration of the absorbing species
- l is the path length of the sample (usually the width of a cuvette, in cm)
Absorbance is measured on a logarithmic scale, representing the ratio of incident light intensity (I₀) to transmitted light intensity (I) as it passes through a sample:
This means that even small changes in concentration can result in detectable changes in absorbance, especially if the system is properly calibrated. Absorbance is a unitless quantity and is a relative measurement, unless the system is specifically calibrated.
Absorbance measurements are wavelength-dependent. Molecules absorb specific wavelengths of light based on their electronic structure, giving rise to characteristic absorption spectra. These spectra serve as molecular fingerprints, allowing for both qualitative identification and quantitative analysis of compounds.
To ensure accurate absorbance measurements, spectrophotometers are typically zeroed or baseline-corrected using a blank (a cuvette containing only the solvent). This corrects for any absorbance by the solvent or cuvette material itself, ensuring only the sample’s contribution is measured. Then the sample is placed in the spectrophotometer and the absorbance spectrum can be measured.
Spectrophotometer vs. Spectrometer
Spectrophotometers are often confused with spectrometers because they can perform similar measurements (absorbance, transmission). They also use similar components such as diffraction gratings and photosensitive detectors. However, there are some key differences between their design and usage.
Spectrometers use a multi-pixel detector array (such as a charge-coupled device) and a fixed diffraction grating to measure a spectrum. This allows them to capture the light intensity of all wavelengths at once, giving a spectrum almost instantaneously. However, the sensitivity of these spectrometer are limited.
Spectrophotometers, on the other hand, generally use a rotating diffraction grating (within a monochromator) and a single-pixel detector. This configuration allows them to scan through a wavelength range one step at a time, building the spectrum gradually. Although this approach is slower, it offers much higher sensitivity, making spectrophotometers better suited for precise, quantitative measurements.
Limitations of Spectrophotometers
While spectrophotometers are extremely valuable in a wide array of applications, there are some useful spectroscopic measurements that are outside of their ability. Principle amongst these are fluorescence measurements. Fluorescence measurements are often used in conjunction with absorption measurements to gain a fuller picture of the electronic configuration of molecules (as absorption is sensitive to excited state of a molecule, but fluorescence is sensitive to its ground state structure). Fluorescence measurements can also be more sensitive for the detection of trace impurities in samples.
Spectrophotometers also struggle to measure absorption of very fluorescent samples. The broadband excitation light may stimulate some fluorescence emission from the sample. This fluorescence is mixed in with the transmitted light, ‘contaminating’ the transmission spectrum.
Both limitations can be overcome by using a device closely related to the spectrophotometer – the spectrofluorometer.
More Resources
Fluorescence Spectroscopy
Fluorescence spectroscopy is used to measure fluorescence. The technique often used together with absorbance spectroscopy. Fluorescence is a type of photoluminescence where light is quickly reemitted from a material after incident photons are absorbed. This is different to phosphorescence where there is a delay between photon absorption and emission. The term fluorescence is often used interchangeably with photoluminescence.
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What is a Photodetector?
A photodetector is a device that can detect light, or more specifically photons. They are classed as optoelectronic devices like photovoltaic devices. This is because they produce an electronic signal which is proportional to the incident optical input.
Read more...