UV-Visible Spectroscopy

BY- K. Sai Manogna (MSIWM014)


Ultraviolet (UV) spectroscopy is an essential physical instrument that utilizes light in the electromagnetic spectrum’s ultraviolet, visible, and near-infrared ranges. The Beer-Lambert law is defined as a linear relationship between absorption, absorber concentration (or absorbing species) in the solution, and path length. Therefore, for a fixed path length, UV-Vis spectroscopy may be used to determine the absorbing species’ concentration. This is a method that is very simple, flexible, quick, precise, and cost-effective. The instrument is called the UV-Vis-NIR Spectrophotometer for ultraviolet-visible (or UV-Vis) spectroscopy. This can be used for the study of liquids, gases, and solids by using radiative energy corresponding to the electromagnetic spectrum’s far and near-ultraviolet (UV), visible (Vis), and near-infrared (NIR) regions. As a result, predetermined wavelengths have been described in these regions: UV ranges in between 300 – 400 nm, whereas visible ranges between 400 – 765 nm, and Near-Infrared ranges in between 765 – 3200 nm. 

Principle: A light beam travels through an object and is determined by the light’s wavelength hitting the detector. The calculated wavelength provides valuable data on the chemical structure and the number of molecules (present in the intensity of the measured signal). Thus, it is possible to obtain both quantitative and qualitative information. Information can be obtained from a wavelength range of 160 to 3500 nm as radiation transmittance, absorbance, or reflectance. Incident power absorption promotes electrons to excited states or anti-bonding orbitals. Photon energy must equal the energy required by electrons to be promoted to the next higher energy state in order for this transition to occur. This method forms the fundamental operating theory of spectroscopy of absorption. Three types of ground-state orbitals can theoretically be involved: 

1. The molecular orbital σ (bonding) 

2. π (bonding) orbital molecular 

3. Atomic Orbital n (non-bonding) 

The anti-bonding orbitals, besides, are: 

  1. The orbital σ* (sigma star) 
  2. The orbital π* (pi star) 

A transition from the s bonding orbital to the σ anti-bonding orbital involving an electron excitation is called the transition from σ to σ*. The excitation of a lone pair electron (non-bonding electron pair) to an anti-bonding π orbital is likewise expressed by π to π*. Electronic transitions that occur due to UV and visible light absorption are: 

  1. from σ to σ*; 
  2. from n to σ*; 
  3. from n to π*; 
  4. from π to π*. 
Ultraviolet-Visible (UV-Vis) Spectroscopy | Analytical Chemistry |  PharmaXChange.info

Fig: Electron transitions in UV-Visible spectroscopy.

Higher energies are included in the transitions s to σ* and n to σ* and thus typically occur in far UV regions or weakly in 180 to 240 nm. Thus, in the UV zone, saturated groups do not demonstrate good absorption. Unsaturated core molecules undergo transitions n to π* and π to π*; these transitions require lower energies and thus occur at longer wavelengths than transitions to anti-bonding orbitals σ*. 

Through the following types of absorption instruments, the UV-Vis spectrum can be recorded: 

  1. Single spectrometer beam 
  2. Spectrometer with double beams 
  3. Simultaneous spectrometer 

All three types of spectrometers have a common light source (mostly tungsten lamps), a smallholder, and a detector. However, besides, a filter can be used to choose one wavelength at a time. This filter is also called a monochromator. A monochromator between the source of light and the specimen is part of the single beam spectrometer. For both wavelengths, the specimen is independently analyzed. The double beam spectrometer uses a single light source, a monochromator, a splitter, and a set of mirrors to direct the beam towards the reference and the sample under investigation. In contrast, a simultaneous spectrometer uses an array of diodes at all wavelengths to simultaneously detect absorbance. The quickest and most potent of the three is this. 

UV-VISIBLE SPECTROPHOTOMETER – Applied Analytical Systems

Fig: Single and double beam UV-Visible spectrometer

Analytical chemistry - UV visible spectroscopy

Fig: Simultaneous UV-Visible spectrometer

Instrumentation: The light source (UV and visible), monochromator (wavelength selector), sample level, and detector are the essential components of a spectrometer. As a light source, a tungsten filament, continuous throughout the UV field, is usually used. Usually, the detector is a photodiode or CCD. To filter light of a specific wavelength, photodiodes go with monochromators to be fed to the detector. The visible lamp must be switched off when tracking the UV spectrum’s absorption, and vice versa. Figure 6 contains a UV-Vis-NIR Spectrometer diagram. 

Components of Instrumental 

A. Source of UV :

1. In its operating wavelength range, the power of the radiating source does not differ. 

2. The continuous UV spectrum is created at low pressures by electrically exciting deuterium or hydrogen. 

3. The UV light generation process involves creating an excited molecular species split into two atomic species and one UV photon. 

4. The emission wavelengths are in the 160 to 375 nm range of both deuterium and hydrogen lamps. 

5. The cuvettes’ content needs to be chosen so that the light incident is not absorbed since this would result in errors in the absorption spectrum obtained. Thus, typically, quartz is used. 

B. Light Source (Visible) 

1. As a visible light source, a tungsten filament lamp is used. 

2. In the 350 to 2500 nm wavelength range, this lamp can produce light. 

3. The energy, i.e., released, is directly proportional to the fourth power of the operating voltage in a tungsten filament lamp. 

4. Thus, a highly stable voltage must be added to the lamp to achieve stable emissions. 

5. By using electronic voltage regulators or constant-voltage transformers, voltage stability is assured. 

6. Tungsten/halogen lamps contain small amounts of iodine, including the tungsten filament, contained within a quartz ‘envelope.’ 

7. The iodine reacts with sublimation-formed gaseous tungsten and creates a WI2 volatile compound. 

8. They decompose when WI2 molecules touch the filament and redeposit tungsten back on the filament. 

9. The tungsten/halogen lamps typically have a lifespan twice the traditional tungsten filament lamp. 

10. Due to their high performance, tungsten/halogen lamps are used in modern spectrophotometers, and their output extends to the UV region as well. 

C. Without Cuvettes

1. The monochromator source is used; light is separated into two sections of equal intensity by a half-mirror splitter before reaching the sample. 

2. One component (or sample beam) passes through the cuvette with the material solution studied in a transparent solvent. 

3. The second beam, or reference beam, passes through a comparable cuvette with only a solvent. 

4. Containers of the reference and sample solution have to be transparent towards the moving beam. 

D. The Detectors 

1. The detector measures the light intensity emitted by the cuvette and sends it to a meter to record and show the values. 

2. The strength of light beams is measured and compared by electronic detectors. 

3. Two detectors have multiple UV-Vis spectrophotometers-a phototube and a photomultiplier tube, and reference and sample beams are simultaneously monitored. 

4. The photomultiplier tube is the detector used widely in UV-Vis instruments. 

5. It requires a photoemissive cathode (when photons strike it, electrons are released from the cathode), several dynodes (when one electron strikes it, a dynode emits several electrons) anode. 

6. The photon incident hits the cathode after it reaches the tube.

7. Furthermore, the cathode releases different electrons, accelerated to the first dynode (whose potential is 90V more positive than cathode). 

8. The first dynode is struck by the electrons, resulting in multiple electrons’ emission with each incident electron. 

9. To create more electrons accelerated towards dynode three, and so on, these electrons are then accelerated towards the second dynode. 

10. At the anode, all the electrons are finally collected. By this time, 106 to 107 electrons had been formed by each initial photon. 

11. Amplified and calculated are the resulting current. Photomultipliers have rapid reaction times and are extremely sensitive to UV and visible radiation. 

12. Photomultipliers, however, are only used for low-power radiation because high-power light will destroy them. 

Schematic diagram of UV-Visible Spectrophotometer | Download Scientific  Diagram

One example of a multichannel photon detector is the linear photodiode array. These detectors will simultaneously measure both elements of a beam of scattered radiation. A linear photodiode array consists of many tiny photodiodes of silicon produced on a single chip of silicon. The number of photodiodes on a chip can vary from 64 to 4096 sensor components, but 1024 photodiodes are the most common. There is a storage capacitor and a switch for each diode. It would help if you sequentially scanned the individual diode- capacitor circuits. 

Photodiode Array Detection in Clinical Applications; Quantitative Analyte  Assay Advantages, Limitations and Disadvantages | IntechOpen

Fig: Photodiode array

Charge-Coupled Devices (CCDs) are like detectors for the diode series, but they consist of an array of photo capacitors instead of diodes. 

The reference beam’s strength should have little to no absorption and is called I0, while the sample beam is called I. Within a short time, the spectrophotometer immediately analyses all wavelength components. In order to assess concentration as well as molecular structure or structural changes, this approach is acceptable. It is also used before and after contact with a substrate or molecule to analyze changes in vibrational and conformational energy levels.

Advantages:

1. The instrument’s precision is the most significant benefit for chemists and astronomers who use UV-VIS spectrometers. 

2. Also, small UV-VIS spectrometers may provide highly accurate readings, which are essential when preparing chemical solutions or recording the celestial body’s movement. 

3. It is quick to use UV-VIS spectrometers. Telescopes are connected to most UV-VIS spectrometers used in astronomy. 

4. In chemistry, most of those users are similar in size to electron microscopes and require the same necessary skills to be used. 

5. Since they are easy to handle, there is little risk of inappropriate use of a UV-VIS spectrometer. 

Disadvantages:

1. The primary downside to using a UV-VIS spectrometer is the time it takes to plan for one to be used. Setup is vital for UV-VIS spectrometers. 

2. The region must be cleared of any visible light, electronic noise, or other external pollutants that could interfere with the spectrometer’s reading. 

3. UV-VIS spectrometers are easy to use and provide precise results if the room has been appropriately prepared ahead of time. 

4. However, even a little bit of outside light or vibration from a small electronic device may interfere with the results you hope to achieve when using a UV-VIS spectrometer if the room has not been appropriately prepared.

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