Each chemical compound has the ability to absorb, reflect and transmit light over an electromagnetic spectrum in wavelengths. When light is transmitted through them a small portion of the light is absorbed. A spectrophotometer helps in both qualitative and quantitative analysis of the sample. As the concentration of a substance increases light absorption increases, and light transmission decreases.
This instrument can be found in chemistry, biochemistry (for enzyme-catalysed reactions), physics, biology, and clinical studies (examining hematology or tissues). It is very useful for scientists to examine various samples without having any surface contact as the specimens are enclosed in a small tube termed a cuvette or in case of the Photopette, estimations are done immediately in the sample vessel without having to transfer it.
A spectrophotometer is a laboratory instrument that is used to measures electromagnetic energy at specific wavelengths of light.
- It can measure the concentration of a known substance in a solution by passing a light through the substance and measuring light intensity as a function of wavelength.
- A spectrophotometer utilizes the properties of light and energy to distinguish colors and determine how much of each color is present in a ray of light
- Using this device we can quickly obtain the spectra of glowing white light on a given specimen thereby measuring the light that is reflected from the specimen.
- In 1940 two scientists Arnold J. Beckman and his colleagues first invented the Beckman DU spectrophotometer at the National Technologies Laboratory (NTL).
A spectrophotometer device helps to measure the light intensity as a function of wavelength. It works by diffracting the light beam into a spectrum of different wavelengths, and then detects the intensities with the help of a charge-coupled device, and representing the results as a graph on the detector and then on the display device.
In a spectrophotometer, the light beam is split by using a prism that diffracts the incident beam into different wavelengths. This device can easily measure the absorption spectrum of a compound, which is the absorption of light by a solution at each wavelength.
A Spectrophotometer is based on the principle of turbidity determination. Turbidity or optical density is the cloudiness of the suspension. The more turbid a suspension, the less light will be transmitted through it. In other words, the amount of light absorbed and scattered is proportional to the mass of cells in the light path. As bacteria grow in a broth, the clear broth becomes turbid. Since the turbidity increases as the number of cells increases, this indicator is used as an indicator of bacterial density in the broth. Turbidity is also useful for standardizing the population densities of bacterial cultures of clinical significance.
Types of Spectrophotometer
There are present different types of Spectrophotometer. They are classified based on their application/uses.
- Visible light spectrophotometer: use visible light from a tungsten lamp; mainly used for routine laboratory practice, particularly the portable and bench-top spectrophotometer models.
- uv vis spectrophotometer: As compared to visible light spectrophotometer, this type of microscope contains a second lamp. UV/Visible spectrophotometer can measure up to 1100 wavelengths. It has different characteristics such as scanning function, user interface, integral printer, and multiple cell setting.
The UV/Visible spectrophotometer is divided into three classes such as;
- Single beam: In a single beam spectrophotometer, a reference standard is used to measure light intensity before and after the sample is loaded. These are typically less expensive and have higher sensitivity.
- Double beam: In a double beam spectrophotometer the beam is split with one beam passing through the standard and the other through the sample to compare intensities. The double beam spectrophotometers tend to be more stable and easier to use.
- Split beam: In this type of spectrophotometer, the light emitted by the same monochromator is split into two beams, one of which reaches the detector directly and the other passes through the sample and reaches the other detector. The advantage of this instrument is that it monitors errors in the light source, but does not eliminate the effects of the reference.
- Near-infrared spectrophotometer: This type of microscope is used to measure the response of a sample when exposed to infrared light. It gives a non-invasive analysis and a quantitative finding with only the least specimen preparation. Those solid samples have a high absorbance, they are mainly monitored by using this Near-infrared spectrophotometer.
- Nuclear Magnetic Resonance spectroscopy: It is used to identify the structure of organic compounds. It gives structural detail of the entire molecule as well as dynamic information of organic reactions.
- Mercury spectrophotometer/analyzer: Used to measure the amount of mercury in water.
- Fluorometers: It is used to measure the fluorescence discharge once the given sample is exposed to a single wavelength of light.
- Atomic absorption spectrophotometer: A flame fumes the water from the specimen causing it to separate into ions. The dissociation leads to changes in the intensity of light as seen by the detector. Hence, help in finding out the concentration of the sample. Atomic absorption spectrophotometer’s high precision analysis is useful in toxicology, environmental testing, and quality control laboratories.
Components of spectrophotometer
A spectrophotometer contain these following components;
- Light Source: The light source is required to generate lights within the spectrophotometer. The material is used in light sources; it should be excited to high energy states by a high voltage electric discharge (or) by electrical heating to serve as excellent radiant energy sources.
- Collimator (lens): This lens points the light to a monochromator or prism from the light source.
- A monochromator (Prisms or Grating): It is used to separate the polychromatic radiation into component wavelength (or) bands of wavelengths. A monochromator determines polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands.
- Wavelength selector (slit): It selects a particular wavelength of light from the splited wavelengths and passes it through the cuvette.
- Transport vessels (cuvettes): The cuvette is used to hold the sample to be studied. It is made up of Quartz.
- A Photosensitive detector and an associated readout system: Most of the detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. Radiation detectors help to produce electronic signals which are equivalent to the transmitter light. These signals require to be turned into a form that is simple to interpret. This is achieved by using amplifiers, Ammeters, Potentiometers and Potentiometric recorders.
Prisms: A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect various wavelengths to a different extent. Two types of Prisms are usually employed in commercial instruments. Namely, 600 cornu quartz prism and 300 Littrow Prism.
Grating: Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible, and infrared regions.
How does a spectrophotometer work?
- The specimen to be studied is placed within the spectrophotometer.
- After that, a light source is shone to generate the light which is then passed through a monochromator where the light splits into each color/individual wavelength.
- These split individual wavelengths hit the subject that is held within a tiny container known as a cuvette. Gently handle the cuvette because the slightest fingerprint can alter the result.
- The light that crosses through the sample is read and interpreted as seen on the output screen.
- The resulting light strikes the photodetector device which compares the intensity of the beam
- Electronic circuits convert the relative currents into linear transmission percentages and/or absorbance/concentration values
- Used for qualitative analysis of a sample.
- Used for quantitative analysis of biochemistry practical for example determine the concentration of an unknown given sample.
- It also used for Enzyme assay.
- To determine the molecular weight of a particular sample, for example amine picrates, ketone compounds, aldehyde, and sugar.
- Used to Identify the impurities.
- To detect the concentration of supplied speciemen.
- To elucidate the structure of Organic compounds.
- To determine the characteristics of a protein.
- To determine the amount of dissolved oxygen in a body of water.
- To analyze the respiratory gas in hospitals.
- Used to detect the Functional group.
- To identify the molecular weight in a particular compound.
- Used to determine the classes of compounds.
- Used in Bial’s test for concentration of pentoses.
- To Estimate the dissolved organic carbon concentration.
- Specific ultraviolet absorbance for metric of aromaticity.
Spectrophotometer calibration is a process by which users find out the light source’s accuracy using a calibration standard. This is an important procedure to make sure that the device functions accurately and the correct measurement is obtained.