Interview Questions on UV-Visible and IR spectroscopy

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Interview Questions on UV-Visible and IR spectroscopy

UV-visible and IR spectroscopy are widely used tools in pharmaceutical development for both qualitative and quantitative analysis. This is the reason why most of the questions are asked about these topics in pharmaceutical analytical or QC interviews. In this post, I will discuss all the questions asked during the interview and their answers. If you want to answer interview questions related to UV and FTIR, then this post is for you.

What is IR spectroscopy?

IR spectroscopy studies the interactions between Infrared radiation and Matter.

It is Electromagnetic radiation.

The range of the infrared region is 12800 ~ 10 cm⁻¹ and can be divided into

• Near-infrared region (12800 ~ 4000 cm⁻¹),
• Mid-infrared region (4000 ~ 400 cm⁻¹) and
• Far-infrared region (50 ~ 1000 cm⁻¹

What is the full form of the FTIR?

Fourier transform infrared

What is FTIR spectroscopy?

The FTIR spectrophotometer is the Michelson Interferometer and it contains

• IR source
• Beam splitter
• Fixed mirror
• Rotating mirror
• Sample compartment and
• Detector

What is the range of the Near-infrared region, Mid-infrared region and Far-infrared region?

• Near-infrared region (12800 ~ 4000 cm⁻¹), 
• Mid-infrared region (4000 ~ 400 cm⁻¹) and 
• Far-infrared region (50 ~ 1000 cm⁻¹

How does infrared radiation interact with the sample?

• Due to internal electronic rearrangement, atoms in the molecule don’t remain fixed at their positions but continuously vibrate at specific frequencies and produce IR radiation
• That vibrational frequency falls in the IR range
• When IR radiation passes through the sample it triggers the vibration of specific molecular bonds  and  specific frequencies of the IR radiation are absorbed
• The absorbed specific frequencies are “missing” from transmitted light

What are the different types of vibration in the molecules?

Symmetric stretching, Antisymmetric stretching and  Bending-like vibrations. Other vibrations are: Rocking, twisting and wagging

What is the Michelson Interferometer?

The Michelson Interferometer contains:

• IR source
• Beam splitter
• Fixed mirror
• Rotating mirror
• Sample compartment and
• Detector

How is the IR test (of a given sample) performed?

• Sample is prepared in KBr or solvent as mentioned in the method of analysis
• First Blank IR (without sample or standard) is performed for the background noise corrections
• Then after standard IR is performed
• Then sample IR is performed &
• Finally, the sample IR spectrum is compared with the standard IR spectrum

What is the procedure to identify a given unknown sample using FTIR?

• Sample is prepared in KBr or solvent as mentioned in the method of analysis
• First Blank IR (without sample or standard) is performed for the background noise corrections
• Then after standard IR is performed
• Then sample IR is performed &
• Finally, the sample IR spectrum is compared with the standard IR spectrum

What is the fingerprint region?

Just like the thumb impression/print of two different persons cannot be the same, similarly the IR spectrum of two different samples cannot be the same.

What are the different applications of FTIR?

It is used for both qualitative and qualitative analysis e.g. functional group identification, structure identification, content tests etc. in the

• Pharmaceutical industries
• Forensic Labs
• Polymer industries
• Foods Industries

Why KBr is used for palate preparation in FTIR?

It gives a transparent palate and secondly, it is readily available in pure for. That is why KBr is used in FTIR.

What is used for the calibration of FTIR?

A polystyrene film reference standard (with valid COA) is used for the calibration of FTIR. The thickness of the Polystyrene film should  be 38µ (micron) or 0.038mm

Why polystyrene film is used for the calibration of FTIR?

The polystyrene coating prevents degradation of the KBr window from moisture content. Secondly is is stable and gives characteristics peaks in FTIR. That is why polystyrene film is used for calibration of FTIR.

What are the peaks of the polystyrene film?

The polystyrene film gives strong FTIR peaks around 2847 – 3068, 1658, 1498, 751, 540cm^-1

What are the differences between KBr mode and ATR mode in FTIR?

In KBr mode pallet is prepped to perform the FTIR whereas in ATR mode direct sample is taken to perform the FTIR.

What are the advantages of FTIR?

• Fast analysis
• Low analysis cost.
• No need of of special skill

What are the weaknesses of the FTIR?

It is more suitable for qualitative analysis (like identification test and functional groups identification) and less suitable for quantitative test (like assay and content test)

Why is water not used in the FTIR?

Water can destroy the KBr window and secondly, water has strong IR peak due to -OH group. That is why water is not used in the FTIR.

What is the working range of the FTIR?

4000 to 600 cm^-1

At what wavenumber carbonyl group give peak in FTIR?

1760 to 1665cm^-1

At what wavenumber hydroxyl group (-OH) gives peak in FTIR?

3500 to 3200 cm^-1

At what wavenumber hydroxyl group (-NH₂) gives peak in FTIR?

3200 to 3600 cm^-1

What is the UV spectroscopy?

UV spectroscopy, or Ultraviolet spectroscopy, is a technique used to measure the absorption of ultraviolet (UV) light by a substance. It involves passing ultraviolet light through a sample and recording how much of the light is absorbed at different wavelengths. This information helps in analyzing the chemical structure and concentration of the substance.

Key elements about UV spectroscopy:

1. Wavelength Range: UV light typically refers to electromagnetic radiation with wavelengths between 200 and 400 nm (nanometers).
2. Absorption: Molecules absorb UV light at specific wavelengths, which is often related to the electronic transitions in the molecule’s structure. When a molecule absorbs UV light, electrons move to higher energy levels.
3. Applications:
   • Qualitative Analysis: Helps identify functional groups or chromophores (parts of a molecule responsible for color) in a compound.
   • Quantitative Analysis: Can be used to determine the concentration of a compound, often through the Beer-Lambert Law, which relates absorbance to concentration.
   • Conformational Analysis: Provides insight into molecular structure, such as the presence of conjugated double bonds.
4. Instrumentation: A UV spectrophotometer is used to measure absorbance or transmittance of UV light through a sample. The results are typically plotted as an absorption spectrum, with wavelength on the x-axis and absorbance on the y-axis.

Which type of solvents are used for sample analysis by UV spectroscopy

Solvents should have a low UV cutoff such as methanol, ethanol, isopropyl alcohol, and water and be able to dissolve the sample.

What is E1% solution?

E1% solution refers to the concentration of a solution in which a substance exhibits an absorbance of 1.0 at a specific wavelength when the solution is at 1% concentration.

Specifically:

• E1%: This stands for the “extinction coefficient” (also called the molar absorptivity) of the substance at a given wavelength, expressed as the absorbance of a 1% (w/v) solution.
• sol: Refers to a solution, indicating the substance is dissolved in a solvent.

For example, if a paracetamol has an E1% sol of 10, it means that a 1% solution of this paracetamol will absorb light at a given wavelength such that the absorbance value is 10 at that wavelength.

What is the UV range?

200 nm to 400 nm

What is the Beer Lambert LAW?

The Beer-Lambert Law, also known as Beer’s Law, is a fundamental principle in spectroscopy that describes the relationship between the absorption of light by a substance and the concentration of that substance in a solution. It is commonly used to measure concentrations of solutes in solutions by analyzing how much light is absorbed as it passes through the solution.

The Beer-Lambert Law is expressed as: A=ε⋅c⋅l

Where:

• A is the absorbance (no units, since it is a ratio),
• ε is the molar absorptivity (or molar absorption coefficient) in units of L·mol⁻¹cm⁻¹
• c is the concentration of the absorbing substance in mol/L,
• l is the path length through which the light passes, typically in cm.

Key Points:

1. Absorbance (A) is directly proportional to both the concentration of the absorbing substance and the path length.
2. Molar absorptivity (ε) is a constant that depends on the substance and the wavelength of light used.
3. The law assumes that the solution is homogeneous and that the absorbance is not too high (i.e., the system is within the linear range of the law).

What is the visible range?

400 nm to 800 nm

Which type of samples are analyzed by UV spectroscopy?

The compounds must have UV absorbance like benzene, toluene and phenol.

What is chromophores?

Chromophores are parts of molecules that are responsible for the absorption of light, specifically in the ultraviolet (UV) or visible spectrum. They contain conjugated systems of alternating single and double bonds, which allow electrons to be excited by light energy. When light strikes a chromophore, the electrons within it are promoted to a higher energy state, which is often what gives the substance its color.

In simpler terms, chromophores are the components of a molecule that absorb certain wavelengths of light, and their absorption leads to the colour we perceive. For example, the chromophore in chlorophyll absorbs light primarily in the red and blue parts of the spectrum, which is why plants appear green (because green light is reflected).

What is Redshift?

Redshift is the change in absorption to a longer wavelength

What is the blue shift?

Blueshift is the change in absorption to a shorter wavelength

What is the effect of solvents on UV Maxima?

The effect of solvents on UV maxima (the wavelengths at which a substance absorbs ultraviolet light) can be significant, as solvents can influence the electronic structure and transition states of a molecule. Here are some key effects:

1. Shift in Absorption Wavelength (Solvatochromism):
   • Solvents can cause a bathochromic shift (red shift) or a hypsochromic shift (blue shift) in the UV maxima depending on the solvent’s properties.
   • Polar solvents: For polar solutes, polar solvents tend to stabilize the excited states, leading to a bathochromic shift (shift to longer wavelengths).
   • Non-polar solvents: Non-polar solvents generally lead to a hypsochromic shift (shift to shorter wavelengths), as they do not stabilize the excited state as much as polar solvents.
2. Solvent-Dependent Absorption:
   • The dipole moment of the solvent can interact with the solute’s electronic structure, particularly influencing transitions that involve charge transfer or polar states.
   • Protic solvents (those capable of hydrogen bonding, like water or alcohols) may cause more significant shifts compared to aprotic solvents, due to their ability to form hydrogen bonds with the solute.
3. Peak Intensity:
   • The solvent can also influence the intensity of UV absorption peaks. Some solvents can broaden or weaken the absorption, while others may sharpen the peaks depending on the solute-solvent interaction and the nature of the molecular transition.
4. Solvent Effects on Transition States:
   • Some solvents can specifically stabilize or destabilize certain molecular orbitals or charge distributions in a molecule, thereby affecting the energy gap between the ground and excited states, which in turn alters the wavelength of absorption maxima.

In summary, solvents can alter the UV maxima of a compound by changing its electronic environment, leading to shifts in absorption wavelengths and sometimes changes in intensity. These shifts are often used in spectroscopic analysis to infer the solvent’s interactions with the solute.

What are the different applications of UV spectroscopy?

The following are the different applications of UV-visible spectrophotometer:

1. Quantitative analysis
2. Qualitative analysis
3. HPLC method development
4. Drug formulations studies

What are the limitations of UV spectroscopy?

It is not specific instrument and not suitable for impure samples

What is the difference between IR spectroscopy and UV spectroscopy?

The difference between IR spectroscopy and UV spectroscopy can be explained based on the following elements:

• Region of the Electromagnetic Spectrum
• Types of Transitions
• What They Measure
• Types of Information Obtained and
• Applications

Region of the Electromagnetic Spectrum:

• IR Spectroscopy: Operates in the infrared region of the electromagnetic spectrum, typically between 4000 cm⁻¹ and 400 cm⁻¹ (or 2.5–25 micrometers in wavelength).
• UV Spectroscopy: Operates in the ultraviolet region of the spectrum, generally between 200 nm and 400 nm in wavelength.

Types of Transitions:

• IR Spectroscopy: Involves the absorption of infrared light by molecules, leading to vibrational transitions. When a molecule absorbs IR light, it causes bonds between atoms to stretch or bend (vibrations).
• UV Spectroscopy: Involves the absorption of ultraviolet light, causing electronic transitions in molecules. It primarily excites electrons from a lower energy molecular orbital to a higher one.

What They Measure:

• IR Spectroscopy: Measures the vibrational frequencies of bonds in molecules. It is particularly useful for identifying functional groups (e.g., alcohols, carbonyls, amines).
• UV Spectroscopy: Measures the absorption of UV light, which is often related to the electronic structure of the molecule, such as conjugated systems (e.g., aromatic rings or double bonds).

Types of Information Obtained:

• IR Spectroscopy: Provides information about molecular vibrations and functional groups. It helps in identifying chemical bonds and structural features of molecules.
• UV Spectroscopy: Provides information about the presence of conjugated π-electron systems or chromophores, as well as the electronic structure of molecules.

Applications:

• IR Spectroscopy: Used to identify functional groups and determine molecular structures.
• UV Spectroscopy: Often used in the analysis of organic compounds with conjugated bonds such as in the study of aromatic compounds. It is also used in qualitative and quantitative analysis.

How IR spectrum can be obtained?

IR spectroscopy or FTIR is a powerful analytical technique used to identify and study compounds by observing how they interact with infrared radiation. When infrared radiation passes through a sample, different types of bonds in the compound absorb energy at characteristic frequencies, corresponding to the vibrational modes of those bonds.

The resulting spectrum is a plot of % transmittance (or absorbance) versus wavenumber, usually expressed in cm⁻¹. The wavenumber represents the frequency of the radiation, and it is directly related to the energy of the vibrations in the molecules. Different bonds (like C-H, N-H, O-H, C=O, etc.) absorb infrared radiation at different wavenumbers, allowing chemists to deduce structural information about the compound.

Key features in an IR spectrum include:

1. Peaks: These correspond to specific bond vibrations absorbing infrared radiation at particular wavenumbers.
2. Fingerprints: The region of the spectrum below ~1500 cm⁻¹, known as the “fingerprint region,” contains complex absorption patterns that are unique to each compound, allowing for precise identification.
3. Functional Group Regions: The higher wavenumber region (typically 4000–1300 cm⁻¹) contains absorption bands that correspond to specific functional groups (like O-H stretching around 3200–3600 cm⁻¹ or C=O stretching around 1700 cm⁻¹).

By analyzing these absorptions, scientists can conclude the functional groups present in the compound and sometimes deduce its molecular structure.

You may also want to check out other articles on my blog, such as:

• Interview questions on GC
• Interview questions on HPLC
• Interview questions on titration

Abbreviations:

• UV: Ultraviolet spectroscopy
• FTIR: Fourier transform spectroscopy