Last updated on February 16, 2022 by
How Does A Spectrophotometer Work
In our post spectrometer vs. spectrophotometer, we explained the difference between the two and how they relate to one another. There, we shared that a spectrophotometer can be any number of instruments that measure light, and that all spectrophotometers use a spectrometer. However, not all spectrometers can be considered spectrophotometers.
The spectrometer plays a major role in measuring the light, and many other analytical instruments contain spectrometers, while the spectrophotometer is the full system that contains a light source along with a means to collect the light for measurement. Furthermore, spectrophotometers are considered a type of photometer.
What Does a Spectrophotometer Measure?
Spectrophotometers employ spectrophotometry to measure the transmittance and absorbance properties of any given material as a function of wavelength, thus determining he concentration of an analyte.
Simply put, spectrophotometers measure light intensity with wavelengths, and can figure out a solution’s concentration using this measurement, based on the Beer-Lambert law (which basically explains that absorbance is going be linear in relation to the concentration—as concentration increases, so does absorbance).
Spectrophotometers also measure electromagnetic intensity at various wavelengths.
Now, because they measure the frequency emitted by the substance that’s being analyzed, spectrometers do not have their own unit to determine the frequency emitted. Instead, measuring units are based on light absorption – wavelength and light intensity. The wavelength of light transmittance or absorbance is measured in nanometers. Because of the small size of the length, the human eye cannot accurately detect it, so machinery is required.
The spectrometer is also capable of providing results on light intensity. However, this requires using multiple complex formulas to calculate the object or sample’s transmittance.
Spectrometers measure a wider range than visible light, which represents just a fraction of the wavelengths of light. The full wavelength of light goes from the gamma-ray (10-5 nanometers) to radio waves (1013 nanometers). Radio waves can be thousands of meters long. The gamma-ray is so small that it is not visible to the human eye
How A Spectrophotometer Works
Let’s first break down all the parts of the instrument, as this makes it easier to understand how everything works together.
- Light source: This is what provides the wavelengths of light at great intensity. The range spans from near-infrared to inside the ultraviolet range, and it includes the visible light spectrum.
- Monochromator: This device acts as the wavelength selector, which filters light from the light source so that only a specific wavelength of light tor range of wavelengths transmits. It comprises a dispersive element, an entrance slit, an exit slit, and mirrors that work together to create a parallel beam of light. The dispersive element can be a prism, or it can be diffraction grating. Both elements are used to separate the light source into specific parts of the spectrum. For instance, when the variable wavelength selector is adjusted, the prism’s position changes so that different wavelengths of light are directed toward the sample compartment that contains the object or sample being analyzed.
- Sample compartment: Here is where you’ll find the transparent tube, also known as a cuvette, that holds the sample you want to analyze, known as the analyte. The wavelengths you select with the assistance of the monochromator pass through the analyte, which are then detected by the photodetector.
- Detector: Light that passes the sample being analyzed hits the detector, which can be any number of types, including photomultiplier tubes (PMTs), silicon photodiodes, InGaAs photodiodes, and photoconductive elements composed of conduction and valence bands. However, the most common detector used today is the PMT. Depending on the target wavelength, a specific detector will be used.
- Display: This component displays the transmittance of the sample. Many models also display the sample’s absorbance, too. Modern spectrophotometers feature a digital display for ease of use.
An important part of the entire instrument is the entrance slit because the size of that slit determines the amount of light that can enter and be measured. This affects not only the speed of the spectrometer’s engine but also the optical resolution. The optical resolution is expressed as the full width at half maximum. Smaller slit sizes translate to a better resolution. The slit can be adjusted to allow for more or less light to enter the spectrometer.
After the light passes through that entry slit, it hits the prism and refracts, then passes through to the sample, which is measured.
Common Types of Spectrophotometers
These devices are either designed as single-beam or double-beam. Single-beam spectrophotometers measure the light intensity before and after the sample is introduced, while double-beam spectrophotometers compare the intensity of light between the reference light path and the sample that’s being measured.
Double-beam models are more accurate because they are not as sensitive to light source fluctuations, but single-beam options have a higher range and are more compact.
Furthermore, spectrophotometers are often categorized based on the specific light they measure within the electromagnetic spectrum, such as the infrared, near-infrared, ultraviolet, or visible range. Depending on the type of light your samples allow to pass through, you’ll want to make sure the device can measure that range.
Infrared spectrometers, sometimes referred to as IR spectrometers, measure vibrations in the interatomic bonds within the sample being tested. When the sample is exposed to infrared wavelengths, the vibrations are measured at different frequencies. This spectrometer can also measure the number of absorbing molecules.
Infrared spectrometers can identify and study chemicals in gas, solid, or liquid form. It is useful for forensic analysis, organic and inorganic chemistry, microelectronics, manufacturing, art history, and various other applications.
Raman spectrometers are most often used in chemistry to provide the structural fingerprint to identify molecules. This type of spectroscopy relies on inelastic scattering of photons. It uses a source of monochromatic light, typically from a laser. Generally, it’s in the visible light, near-infrared, or near-ultraviolet spectrum, though it’s also possible to use x-rays.
The laser interacts with excitations within the sample, which shifts the energy either up or down. That shift provides information about the vibrational modes, similar to the information infrared spectroscopy offers.
UV-Visible spectroscopy exposes the sample to ultraviolet light, which excites the electrons upon absorbance of the light energy. The absorbance is measured based on how excited the electrons become. This type of spectroscopy is commonly used to research the chemical bonding of molecules in the sample material.
Near IR spectroscopy is based on the absorption of electromagnetic radiation at wavelengths from 780 to 2,400 nanometers. The light interacts with the sample and then the detector measures the transmittance and absorbance. Near IR spectroscopy has a wide range of applications, including, neonatal research, blood sugar, functional neuroimaging, urology, ergonomics, atmospheric chemistry, and more.
X-Ray spectrometers excite the inner electrons of the sample. When the excited electrons fall into the empty space generated as a result of energy absorption, x-rays are procured.
Regardless of the type, make, or model of spectrophotometer your lab requires, Excedr can help you get what you need.
Leasing vs. Buying A Spectrophotometer
Spectrophotometers are expensive pieces of equipment. No matter how established your lab is, purchasing either new or used can take a big bite out of your budget.
Leasing your lab equipment, on the other hand, enables you to acquire equipment you need at a much more affordable price point up front.
And because Excedr takes care of all the necessary equipment maintenance and upkeep, you can focus more of your time and effort on the tasks that really matter.