How Does a Modern UV-Vis Spectrophotometer Work?

The UV-Visible spectrophotometer is developed to measure the quantity and quality of samples utilizing Ultraviolet-visible light varying with the wavelength range 200nm to 900nm. 

In 1814 Fraunhofer Gesellschaft, invented a spectroscope to measure sunlight and he later discovered 574 dark fixed lines popularly known as Fraunhofer lines. 

In 1821 he developed diffraction grating to separate light from the sun. After 40 years, David Rittenhouse invented the first man-made diffraction grating.

After a century later in 1941, Arnold O Beckman introduced the first commercial UV-Visible spectrophotometer to determine the quantity and quality of the samples. 

In the first developed instrument, a quartz prism was used to separate light source (from the tungsten lamp) into the absorption spectrum and a phototube is used in the place of the modern photodiode as a detector. 

UV-Vis spectrophotometer measures the intensity of light transmitted and absorption of the samples that represent the properties of the sample.

Even though the optical principle of modern UV-Vis spectrophotometer has not changed much, recent developments in the optical components have made modern UV-Vis spectrophotometer more robust, faster, more versatile, and compact.

UV/Vis spectroscopy is the widely used analytical technique in various scientific researches such as analytical chemistry, bacterial culturing, nucleic acid purity, quality control in food and beverage industries, drug identification, pharmaceuticals, and more. 

Here we discuss, how UV- Visible spectrophotometer works, its strength, and its limitations over various applications.

What is UV-Vis spectroscopy?

UV-Vis spectroscopy is an analytical tool that measures the absorption or transmission of light through the sample in comparison to a reference or blank. Every substance has a unique way of light absorption. 

From this technique, the properties, concentration, and composition of the sample can be determined. The spectroscopy technique completely relies on the light source, it is a must to understand the properties of light.

Light is electromagnetic radiation that travels with a certain amount of energy. The amount of energy the light travels is inversely proportional to wavelength (shorter the wavelength more the energy the light carries and vice versa). 

Absorption of light is detected in a substance when there is a change in a certain amount of energy to promote electrons from one state to the other. Different substances have different electron bonding and hence absorption of light occurs at different wavelengths. 

Only a spectrum of light with a wavelength ranging from 380nm (violet) to 780nm(red) is visible to the human eye. UV light has wavelengths shorter than visible light. 

This concept of light described in wavelength corresponds to the absorbance of light by the substance is used in the UV spectrophotometer to understand different substance properties and analyze them quantitatively and qualitatively.

How does a UV-Vis spectrophotometer work?

We do understand the basic concept behind light and why it is used UV-Vis spectrophotometer. For a better understanding of how does UV-Vis spectrophotometer works? we consider only the main components of the spectrophotometer as shown in the below figure.

Light source

As a spectrophotometer is a light-based technique, it is a must to choose a steady and bright light source. Basically, the light source of the spectrophotometer should include,

• a) High degree and uniform brightness across the wavelength range.
• b) stable over time,
• c) long service life, and
• d) low cost.

Achieving all these requirements in a single light source is not possible. Most of the spectrophotometers use two light sources halogen tungsten lamps for visible range and deuterium lamps for ultraviolet range with respect to the wavelength setting in order to achieve both high degrees of brightness and uniform brightness distribution.  

Practically the switchover between two light sources occurs in the wavelength scan between 300 and 350nm as the light emission for the light source is similar in this wavelength range and hence the transition can be made smoothly. 

Switching between light sources also has a few more advantages such as reduction of incident light to the monochromator and reducing the amount of stray light.

Some spectrophotometer also uses Xenon flash lamp that is suitable for target analysis. Xenon lamp is used as a high-intensity light source for both UV and visible ranges but the cost of the xenon lamp is higher and less stable compared to halogen tungsten and deuterium lamps.

Wavelength selector

The light source that releases a wide range of wavelengths. It is required to select a certain wavelength that suits the sample for examination and analyte for detection. The following are the different methods used for wavelength selection.

• Monochromators- A light with narrow bandwidth or light of single color can be obtained with the help of a monochromator. Diffraction gratings used in the monochromator can be rotated to choose the desired wavelength. 
  
  There are two parts in the diffraction grating are blaze angle and the groove frequency. The groove frequency is generally measured as the number of grooves per mm. Better optical resolution is obtained with higher groove frequency but a narrow wavelength range.  
  
  Lower the groove frequency wider the wavelength range but poor optical resolution. Typically 1200 grooves/mm are used in the UV-Vis spectrophotometer.
  
  There are two different types of diffraction gratings. One is holographic grating and the other is ruled grating. The results and accuracy of spectroscopy measurements are depending on the diffraction gratings and the optical setup. 
  
  Holographic grating provides a uniform spectral response but lower efficiency. Ruled gratings are simple and cheap but produce more stray light due to physical imperfections and hence holographic gratings are chosen for better results compared to that of the ruled gratings.
• Absorption filters – Absorption filters are generally made up of color filter glass or pigmented gelatin resins or plastic-coated glass. This filter operates based on the absorption of a specific wavelength of light.
• Interference filters – Interference filters are also referred to as the dichroic filters that operate with the reflection of one or more spectral bands and transmit others. These filters use thin layers of dielectric material and eliminate the undesirable wavelength by destructive interference. 
• Cutoff filters – These filters are developed with interference filters. These filters allow certain wavelengths that are either below or above a specific wavelength.
• Bandpass filters –It is the combination of short pass and long pass filters that allow a certain range of wavelengths to pass through.

Generally, monochromators are used in the spectrophotometer as a wavelength selector due to their versatility. In order to improve the signal-to-noise ratio and for precise measurements filters are often used with the monochromators.

Sample analysis

After selecting a certain range of wavelengths, the light is then passed through the sample. Measuring a reference sample is referred to as a “blank sample” in a cuvette 

Generally, quartz holders are used as it is transparent to the majority of UV light. But plastic and glass cuvettes are inappropriate as it absorbs the majority of UV light. 

Even air also absorbs certain wavelengths of light shorter than 200nm because of the presence of molecular oxygen in the air. An expensive setup is required for measurements less than 200 nm. In the case of DNA  and RNA analyses, cuvette is not required in the spectrophotometer.

Detection

It is important to convert the light into a readable electronic signal after light passes through the sample and hence there comes the role of detectors. There are different types of detectors used in the spectrophotometer,

Photomultiplier Tubes (PMT): It is a commonly used detector in UV-Visible spectrophotometers. It works based on the principle of the photoelectric effect. It initially releases an electron on exposure to light subsequently multiple electrons are released to generate current. PMT  detectors are efficient and useful while detecting low levels of light.

Silicon photodiodes: Silicon photodiodes when exposed to light produce an electric current proportional to the light intensity.

CCD ( Charge-Coupled Device) is a semiconductor device, that acts as a detector and converts light intensity into electrical energy.

Once the light intensity is converted to the electric signal, the signal is recognized as an output with the help of a computer screen with specific software.

Conclusion

There are many UV/Vis spectrophotometer applications in scientific research such as bacterial culture, hemoglobin concentrations cancer research, beverage analysis, and wide applications in quality control. 

It is important to know the concept and basic working of the spectrophotometer to choose the right spectrophotometer with the optimum specifications that are required for the research.