LC–FTIR, LC–NMR, CE–MS and HPTLC applications MCQs With Answer

Introduction: This quiz set focuses on advanced hyphenated and planar chromatography techniques—LC–FTIR, LC–NMR, CE–MS and HPTLC—commonly encountered in M.Pharm spectral analysis courses. It is designed to test both conceptual understanding and practical aspects: instrument interfaces, solvent and buffer selection, sensitivity challenges, data interpretation, and typical applications such as natural product profiling, impurity identification and method development. Questions emphasize real-world considerations (e.g., solvent suppression in LC–NMR, sheath vs sheathless CE–MS interfaces, solvent transparency for LC–FTIR, and derivatization/densitometry in HPTLC). Answers are provided to help self-assess and prepare for examinations and laboratory work.

Q1. Which of the following best describes a major challenge when coupling LC to FTIR for on-line detection?

• Requirement for non-volatile salts in the mobile phase
• High background absorption from common LC solvents in the infrared region
• Incompatibility of FTIR detectors with chromatographic flow rates
• Inability of FTIR to detect functional groups

Correct Answer: High background absorption from common LC solvents in the infrared region

Q2. In LC–NMR, what is the primary advantage of using the stop-flow (loop-and-wait) approach compared with continuous-flow acquisition?

• It eliminates the need for solvent suppression
• It allows signal averaging on a single chromatographic peak to improve sensitivity
• It increases chromatographic resolution by slowing elution
• It permits direct coupling to MS without loss of sample

Correct Answer: It allows signal averaging on a single chromatographic peak to improve sensitivity

Q3. Which interface is commonly used to couple capillary electrophoresis (CE) to mass spectrometry (MS) and offers higher robustness though slightly less sensitivity?

• Sheathless porous-tip interface
• Membrane desolvation interface
• Sheath-flow (coaxial sheath liquid) electrospray interface
• Direct atmospheric pressure chemical ionization (APCI) interface

Correct Answer: Sheath-flow (coaxial sheath liquid) electrospray interface

Q4. Which statement about HPTLC (high-performance thin-layer chromatography) is most accurate?

• HPTLC cannot be used for quantitative analysis
• HPTLC routinely requires vacuum MALDI for detection
• HPTLC allows parallel analysis of many samples with post-chromatographic derivatization and densitometric quantitation
• HPTLC uses only aqueous mobile phases

Correct Answer: HPTLC allows parallel analysis of many samples with post-chromatographic derivatization and densitometric quantitation

Q5. For LC–FTIR analysis, which strategy is commonly employed to minimize solvent interference in the IR spectrum?

• Use of non-volatile buffers such as phosphate
• Use of very long pathlength flow cells
• Use of solvent-elimination interfaces or ATR (attenuated total reflectance) sampling to reduce solvent background
• Replacing FTIR with UV detection

Correct Answer: Use of solvent-elimination interfaces or ATR (attenuated total reflectance) sampling to reduce solvent background

Q6. In LC–NMR, which solvent approach simplifies field-frequency locking and reduces the need for continuous solvent suppression?

• Use of strong non-volatile buffers
• Use of deuterated mobile phases or a deuterium lock stream
• Use of high-salt aqueous buffers
• Use of pure methanol without any deuteration

Correct Answer: Use of deuterated mobile phases or a deuterium lock stream

Q7. What is a primary advantage of a sheathless CE–MS interface compared to a sheath-flow interface?

• Greater mechanical robustness and easier maintenance
• Higher sensitivity due to lower dilution of analyte by sheath liquid
• Ability to use non-volatile high-concentration buffers
• Higher flow rates for compatibility with APCI

Correct Answer: Higher sensitivity due to lower dilution of analyte by sheath liquid

Q8. Which mobile phase component is generally problematic for CE–MS coupling because it suppresses ionization in electrospray?

• Volatile ammonium acetate
• Tris(hydroxymethyl)aminomethane (Tris) at high concentration
• Formic acid at low concentration
• Acetonitrile

Correct Answer: Tris(hydroxymethyl)aminomethane (Tris) at high concentration

Q9. Which HPTLC step improves visibility and detection of compounds that are UV-inactive?

• Shortening the chromatographic run time
• Post-chromatographic derivatization using reagents such as anisaldehyde, vanillin, or Dragendorff
• Increasing plate thickness to 500 µm
• Using only UV 254 nm illumination

Correct Answer: Post-chromatographic derivatization using reagents such as anisaldehyde, vanillin, or Dragendorff

Q10. When interpreting LC–FTIR spectra for structure elucidation, which information is most directly obtained?

• Molecular weight and isotopic pattern
• Functional group vibrations such as carbonyl, OH, NH stretches
• Exact 3D conformation and stereochemistry
• Proton-proton coupling constants

Correct Answer: Functional group vibrations such as carbonyl, OH, NH stretches

Q11. In LC–NMR, which hardware option significantly increases sensitivity for small sample amounts?

• Using a standard 5 mm wide-bore probe
• Using a microcoil (capillary) NMR probe or cryoprobe
• Reducing the magnetic field strength
• Operating without any shimming

Correct Answer: Using a microcoil (capillary) NMR probe or cryoprobe

Q12. Which application is CE–MS particularly well suited for in pharmaceutical analysis?

• High-volume preparative isolations of large hydrophobic drugs
• Separation and mass characterization of charged small molecules, peptides, and metabolites at low sample volumes
• Quantitative analysis of nonpolar lipids without separation
• Direct solid sample analysis of tablets without dissolution

Correct Answer: Separation and mass characterization of charged small molecules, peptides, and metabolites at low sample volumes

Q13. For HPTLC quantitative analysis, which instrumental technique is most commonly used to measure spot intensity?

• Orbital shaking
• Densitometric scanning at selected wavelengths
• Gas-phase ionization
• Infrared imaging only

Correct Answer: Densitometric scanning at selected wavelengths

Q14. In LC–FTIR, the solvent-elimination interface (SEI) is used so that analytes reach the FTIR cell as what form?

• In their fully protonated ionic forms in aqueous stream
• As a dry or concentrated deposit after solvent removal to reduce background absorption
• Dissolved in a high-absorption IR solvent
• Complexed with metal ions to enhance IR signals

Correct Answer: As a dry or concentrated deposit after solvent removal to reduce background absorption

Q15. Which NMR technique is commonly applied on-line in LC–NMR to suppress the strong solvent signal from water or organic solvents?

• Fourier transform only without pulse sequences
• Solvent suppression sequences such as presaturation, WATERGATE or WET
• High-power broadband decoupling during acquisition only
• Use of non-uniform sampling without suppression

Correct Answer: Solvent suppression sequences such as presaturation, WATERGATE or WET

Q16. What is an important validation parameter to assess when transferring an HPTLC method for quantitation of an herbal marker to routine quality control?

• Only the color of the derivatization reagent
• Linearity, precision (repeatability), accuracy (recovery), LOD/LOQ and robustness
• Number of developers used in the lab the first week
• Only the brand of silica gel plates

Correct Answer: Linearity, precision (repeatability), accuracy (recovery), LOD/LOQ and robustness

Q17. Which factor most improves CE separation selectivity for positional isomers of small charged drugs?

• Using a very high concentration of non-volatile salts
• Modifying the capillary wall with appropriate static or dynamic coatings and optimizing buffer pH and ionic strength
• Always operating at pH 7 regardless of analyte pKa
• Replacing the capillary with an HPLC column

Correct Answer: Modifying the capillary wall with appropriate static or dynamic coatings and optimizing buffer pH and ionic strength

Q18. In CE–MS, what common mass spectral artifact must analysts be aware of when interpreting peaks for small molecules?

• Absence of isotope patterns in organic molecules
• Formation of adducts (e.g., [M+Na]+, [M+K]+) and in-source fragmentations that can complicate identification
• Complete lack of ionization for all analytes
• Only singly charged ions are observed for peptides

Correct Answer: Formation of adducts (e.g., [M+Na]+, [M+K]+) and in-source fragmentations that can complicate identification

Q19. Which advantage does on-line LC–NMR provide over off-line NMR of isolated fractions?

• Eliminates the need for any purification prior to analysis
• Provides direct structural information of chromatographic peaks in situ, saving time and reducing sample handling/artifacts
• Makes mass spectrometric data unnecessary
• Always has better sensitivity than off-line NMR

Correct Answer: Provides direct structural information of chromatographic peaks in situ, saving time and reducing sample handling/artifacts

Q20. For HPTLC analysis of complex herbal extracts, which workflow element most improves component identification and confirmation?

• Single-wavelength UV visualization only
• Combination of multi-wavelength densitometry, specific derivatization reagents and comparison with authentic standards or hyphenated MS detection (HPTLC–MS)
• Using the thickest possible stationary phase to trap compounds
• Avoiding any reference standards to prevent bias

Correct Answer: Combination of multi-wavelength densitometry, specific derivatization reagents and comparison with authentic standards or hyphenated MS detection (HPTLC–MS)

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