What is a Spectrophotometer? What Does It Measure?
Jump to: How Spectrophotometers Work | Absorbance Measurements | Spectrophotometer vs. Spectrometer | Spectrophotometer Limitations
A spectrophotometer is a piece of spectroscopy equipment that can measure the amount of light absorbed by a sample. This measurement can be useful in many research applications:
To identify materials by mapping molecular absorption profiles.
- To work out solute concentrations in solutions.
- For detecting trace impurities in samples
- Following the progress of chemical reactions.
There are four important components within a spectrophotometer: a light source and a monochromator (combined to make a tunable light source), a sample holder and a detector. Light from a broadband light source is dispersed by a monochromator. This dispersed light is directed towards the sample through an adjustable slit. This allows a fraction of disperse light though with a narrow wavelength range. Using a rotating diffraction grating, the monochromator can select which wavelengths are transmitted through the sample.
Some wavelengths of light will be more strongly absorbed by the sample than others, and this information can give you information about its molecular structure. The detector measures the intensity of transmitted light, so by measuring the light intensity transmitted at a range of wavelengths, the spectrophotometer can build an absorbance spectrum.
How Does A Spectrophotometer Work?
The four essential components to a spectrophotometer are as follows:
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A Light Source
Spectrophotometers require a broadband (white) light source e.g. halogen bulb, arc lamp or an LED light source, to measure an absorption spectrum.
However spectrophotometers differ from spectrometers as the split broadband light into component wavelengths before measurement. Spectrometers use CCD arrays in their detector components, which measure across the wavelength range entire wavelength range at once. This is a fast measurement, but its measurement precision and sensitivity is limited.
Spectrophotometers often use a strong broadband light source in combination with a monochromator, to create a tunable light source. The sample is only exposed to a small portion of light at a time, and the rotating diffraction grating within the monochromator allows users to choose or vary this excitation wavelength. Combining a variable light source with a single pixel detector, spectrophotometers can measure smaller signals with higher accuracy and resolution compared to spectrometers.
Alternative to this, you can use a monochromatic light source (i.e. a laser) with your spectrophotometer, but you will only be able to measure absorption at a specific wavelength. This is suitable for some absorbance measurement such as OD600 measurements used in microbiological studies.
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A Sample Chamber
The placement and alignment of your sample within the spectrophotomer is important. The sample chamber is designed to ensure that sample positioning is consistent between experiments, improving reliability and repeatability. Spectrophotometers often have specific sample holders for different materials e.g. for a thin film or an optical cuvette.
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Light Intensity Detector
Once light has passed through the sample, its intensity needs to be quantified, using a photodetector. This photodetector will convert incident light into an electrical signal proportional to the incident light intensity. 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.
To improve accuracy and sensitivity, spectrophotometers use a single pixel detector. These have a greater measurement area that other alternatives, such as CCD arrays used in spectrometers, allowing them to measure lower signal changes improving sensitivity. However, they cannot distinguish between different wavelengths of light. This is why single pixel detectors must be combined with a monochromator, to separate light before measurement.
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Monochromators
The light from the sample needs to be split into its component wavelengths as the detectors cannot distinguish between different wavelengths. This dispersion is achieved using a monochromator, which consists of an entrance slit, a diffraction grating (or less commonly a prism) and focussing mirrors. The diffraction grating disperses light, and rotates to direct a different portion of the spectrum towards an exit slit. This mechanism allows you to vary the output wavelength easily and reliably.
Measuring Absorbance Using A Spectrophotometer
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 absorbing 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:
Absorbance is a unitless quantity and is a relative measurement. But one conclusion that can be drawn from these two equations, is that even small changes in concentration can result in singnificant changes in absorbance.
Absorbance measurements are wavelength-dependent. Molecules absorb specific wavelengths of light based on their electronic structure, giving rise to characteristic absorption spectra for every material. 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 is measured.
Spectrophotometer vs. Spectrometer
Spectrophotometers are often confused with spectrometers and can be used for measurements (e.g. absorbance, transmission). They also have similar components: diffraction gratings, photosensitive detectors. However, there are some key differences between spectrophotometers and spectrometers.
Spectrometers use a multi-pixel detector array (such as a charge-coupled device) as a detector. A fixed diffraction grating within the spectrometer disperses incoming light, and directs all wavelengths towards the CCD. This allows a spectrometer to capture the light intensity of all wavelengths at once, giving a spectrum almost instantaneously. However, the sensitivity of spectrometers are limited.
Spectrophotometers, on the other hand, generally use a rotating diffraction grating (within a monochromator separate to the detector) and a single-pixel detector. This configuration allows these instruments 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 valuable in a wide array of applications, there are some measurements that are outside of their ability. Principle amongst these are fluorescence measurements. Fluorescence measurements are often used alongside absorbance measurements to gain a better picture of an molecule's electronic configuration. Absorption is sensitive to excited state of a molecule, but fluorescence is sensitive to its ground state structure. Fluorescence measurements are also more useful for detecting trace impurities in samples.
Furthermore, spectrophotometers 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, which includes monochromators, detectors and light sources aligned to measure absorbance and fluorescence measurements with one instrument.
Monochromator
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