LC-NMR hyphenation MCQs With Answer

Introduction: LC‑NMR hyphenation combines liquid chromatography’s separation power with nuclear magnetic resonance’s structural elucidation, offering a powerful tool for M.Pharm students engaged in drug analysis, impurity profiling and natural product characterization. This blog presents focused multiple‑choice questions that probe practical and theoretical aspects of LC‑NMR coupling: interfaces and flow cells, solvent suppression strategies, sensitivity and sample concentration techniques, pulse sequence choices, stop‑flow vs continuous modes, and troubleshooting common hyphenation problems. Questions are designed to deepen understanding of instrument configuration, method development and data interpretation so students can apply LC‑NMR effectively in pharmaceutical research and quality control.

Q1. What is the primary challenge when coupling reversed‑phase LC directly to NMR without modification?

• Excessive sample dilution leading to low NMR sensitivity
• Incompatibility of mobile phase pH with NMR detection
• Magnet quenching due to solvent polarity
• Inability to detect UV‑inactive compounds

Correct Answer: Excessive sample dilution leading to low NMR sensitivity

Q2. Which hardware is most commonly used to maintain magnetic field homogeneity for flowing samples in LC‑NMR?

• Standard 5 mm NMR tube
• Flow NMR probe with glass or PEEK flow cell
• Capillary LC column directly placed in the magnet bore
• Inline UV detector placed near the magnet

Correct Answer: Flow NMR probe with glass or PEEK flow cell

Q3. In online LC‑NMR, what is the main purpose of using deuterated solvents or deuterium locking techniques?

• To increase chromatographic resolution
• To provide a lock signal for magnetic field stability and minimize solvent peak complications
• To allow detection of halogenated compounds
• To improve mass spectrometry compatibility

Correct Answer: To provide a lock signal for magnetic field stability and minimize solvent peak complications

Q4. Which of the following solvent suppression techniques is commonly used in LC‑NMR to reduce the large mobile phase signal?

• Presaturation or selective presaturation
• Cryogenic cooling only
• High‑performance liquid chromatography (HPLC) gradient
• Using nonpolar stationary phases

Correct Answer: Presaturation or selective presaturation

Q5. What is the advantage of stop‑flow LC‑NMR compared with continuous flow LC‑NMR?

• It eliminates the need for solvent suppression
• It enables accumulation of scans for improved sensitivity on a single chromatographic fraction
• It allows simultaneous MS and NMR acquisition
• It increases chromatographic throughput

Correct Answer: It enables accumulation of scans for improved sensitivity on a single chromatographic fraction

Q6. Which probe design improves sensitivity for low‑mass analytes in LC‑NMR?

• Standard room‑temperature broad‑bore probe
• Cryoprobe or microcoil flow probe
• High‑resolution solid‑state probe
• Ultrawideband passive probe

Correct Answer: Cryoprobe or microcoil flow probe

Q7. When hyphenating LC with NMR, why is sample concentration often performed offline using SPE (solid‑phase extraction)?

• To remove salts that interfere with MS only
• To pre‑concentrate analytes and replace the mobile phase with an NMR‑friendly solvent, improving sensitivity
• To change analyte pKa permanently
• To enable faster chromatographic separations

Correct Answer: To pre‑concentrate analytes and replace the mobile phase with an NMR‑friendly solvent, improving sensitivity

Q8. Which of the following pulse sequences is especially useful in LC‑NMR for observing 1H spectra while suppressing solvent signals?

• INEPT for heteronuclear 13C detection
• Presaturation or WATERGATE/WET solvent suppression sequences
• Noesy‑flip for quantitative 1H
• TOCSY for 13C coupling networks

Correct Answer: Presaturation or WATERGATE/WET solvent suppression sequences

Q9. What is the role of an interface such as a flow splitter or stop‑flow valve in LC‑NMR systems?

• To increase column backpressure
• To divert part or all of the eluent to the NMR flow cell or to collection for offline concentration
• To convert UV‑active compounds to NMR‑active species
• To change the magnet temperature

Correct Answer: To divert part or all of the eluent to the NMR flow cell or to collection for offline concentration

Q10. Why is 13C detection less frequently used in direct online LC‑NMR compared with 1H detection?

• Because 13C nuclei cannot be detected in flow cells
• Because 13C has lower natural abundance and sensitivity, requiring much longer acquisition times
• Because 13C spectra are overwhelmed by solvent 1H signals
• Because 13C detection requires deuterated stationary phases

Correct Answer: Because 13C has lower natural abundance and sensitivity, requiring much longer acquisition times

Q11. In LC‑NMR, what is a major benefit of using stop‑flow with multiple transient accumulations compared to a single long acquisition?

• It avoids the need for mobile phase degassing
• It allows verification of signal stability and correction for baseline drift between accumulations
• It reduces the amount of solvent used in LC
• It eliminates the need for shimming

Correct Answer: It allows verification of signal stability and correction for baseline drift between accumulations

Q12. Which statement best describes why gradient HPLC mobile phases complicate LC‑NMR?

• Gradient elution causes variable solvent composition that changes 1H background and makes solvent suppression and field locking more difficult
• Gradient elution results in complete sample decomposition before NMR detection
• Gradient elution prevents use of deuterated solvents
• Gradient elution reduces chromatographic resolution irreversibly

Correct Answer: Gradient elution causes variable solvent composition that changes 1H background and makes solvent suppression and field locking more difficult

Q13. Which practice improves quantitative reliability of LC‑NMR measurements?

• Using presaturation at high power for all peaks
• Adding an internal standard with known concentration that is NMR‑visible and chromatographically separated
• Using nondeuterated water as the mobile phase without suppression
• Maximizing flow rate to reduce residence time

Correct Answer: Adding an internal standard with known concentration that is NMR‑visible and chromatographically separated

Q14. Which of the following is a common limitation of LC‑NMR compared with LC‑MS?

• LC‑NMR cannot provide structural information
• LC‑NMR has much lower sensitivity and typically requires higher sample amounts
• LC‑NMR is incompatible with reversed‑phase separations
• LC‑NMR always requires cryogenic cooling

Correct Answer: LC‑NMR has much lower sensitivity and typically requires higher sample amounts

Q15. When designing an LC‑NMR experiment for a mixture of trace impurities, which strategy best increases the chance of obtaining usable NMR spectra?

• Use a shorter column to decrease peak width
• Preconcentrate impurities by fraction collection and offline evaporation into an NMR‑friendly solvent or use SPE trapping
• Run LC at the highest possible flow rate
• Use only 13C detection to avoid solvent issues

Correct Answer: Preconcentrate impurities by fraction collection and offline evaporation into an NMR‑friendly solvent or use SPE trapping

Q16. In hyphenated LC‑NMR, what is a microcoil probe particularly useful for?

• Improving resolution for very large macromolecules only
• Enhancing mass sensitivity for very small sample volumes and low concentration analytes
• Reducing the need for solvent suppression
• Allowing direct injection of solid samples into the magnet

Correct Answer: Enhancing mass sensitivity for very small sample volumes and low concentration analytes

Q17. Why is shimming more challenging in LC‑NMR compared with static tube NMR?

• Because flowing liquids always have lower viscosity
• Because moving interfaces, variable solvent composition, and small flow cells produce dynamic magnetic susceptibility changes that disturb field homogeneity
• Because LC columns generate magnetic fields that interfere with shims
• Because flow NMR probes do not allow shim adjustment

Correct Answer: Because moving interfaces, variable solvent composition, and small flow cells produce dynamic magnetic susceptibility changes that disturb field homogeneity

Q18. For structural elucidation using LC‑NMR, which 2D experiment is most informative for establishing proton connectivity within an eluted fraction?

• 13C DEPT experiment
• 1H‑1H COSY or TOCSY
• 31P NMR only
• Solid‑state CP/MAS experiment

Correct Answer: 1H‑1H COSY or TOCSY

Q19. What is one advantage of using partially deuterated mobile phases (e.g., D2O/organic) in LC‑NMR?

• They eliminate the need for shimming entirely
• They provide sufficient lock signal while reducing proton background to facilitate solvent suppression
• They increase column lifetime
• They improve MS sensitivity when coupling to LC‑MS

Correct Answer: They provide sufficient lock signal while reducing proton background to facilitate solvent suppression

Q20. When combining LC‑NMR with LC‑MS sequentially, what is a typical workflow to maximize information from a single run?

• Divert the entire eluent to NMR first, then to MS without splitting
• Use a post‑column splitter to send part of the flow to MS for mass information and part to the NMR flow cell (or collect fractions for offline NMR) for structural data
• Perform MS only and infer NMR spectra computationally
• Run LC twice, once for MS and once for NMR, with identical chromatographic conditions always

Correct Answer: Use a post‑column splitter to send part of the flow to MS for mass information and part to the NMR flow cell (or collect fractions for offline NMR) for structural data

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