NOESY and COSY techniques MCQs With Answer

Introduction

This quiz set on NOESY and COSY techniques is designed specifically for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). It focuses on practical and theoretical aspects of two-dimensional NMR experiments used widely in structural elucidation of pharmaceutical molecules. Questions cover principles of through-bond (COSY) and through-space (NOESY) correlations, pulse sequences, mixing-time effects, interpretation of cross-peaks, artifacts, and applications in stereochemistry and drug research. The items emphasize conceptual understanding and experimental considerations such as relaxation, spin diffusion, solvent suppression and appropriate parameter choices so you can apply NOESY/COSY knowledge to real-world structure-determination problems.

Q1. What fundamental interaction does a COSY (Correlation Spectroscopy) experiment primarily detect?

• Dipolar through-space interactions between nearby protons
• Through-bond scalar (J) coupling correlations between nuclei
• Chemical exchange processes between conformers
• Cross-relaxation rates proportional to 1/r6

Correct Answer: Through-bond scalar (J) coupling correlations between nuclei

Q2. What is the primary physical mechanism responsible for cross-peaks observed in a NOESY experiment?

• Scalar (J) coupling mediated coherence transfer
• Dipolar Nuclear Overhauser Effect (through-space cross-relaxation)
• Spin-lock driven isotropic mixing
• Exchange of chemically equivalent sites

Correct Answer: Dipolar Nuclear Overhauser Effect (through-space cross-relaxation)

Q3. How does the NOESY mixing time ™ influence the spectrum and what is the practical consideration?

• Longer tm reduces sensitivity and should always be maximized
• tm controls NOE buildup; it must be optimized to observe true spatial proximities while avoiding spin diffusion
• tm only affects solvent suppression and is unrelated to cross-peak intensity
• tm determines J-coupling evolution and should match 1/J values

Correct Answer: tm controls NOE buildup; it must be optimized to observe true spatial proximities while avoiding spin diffusion

Q4. Why is ROESY sometimes preferred over NOESY for medium-sized molecules (e.g., 1–10 kDa)?

• ROESY generates through-bond correlations unlike NOESY
• ROESY avoids the near-zero or negative NOE regime by using rotating-frame cross-relaxation, giving reliable positive cross-peaks for intermediate correlation times
• ROESY requires no mixing time optimization
• ROESY suppresses J-coupling completely and shows only chemical exchange

Correct Answer: ROESY avoids the near-zero or negative NOE regime by using rotating-frame cross-relaxation, giving reliable positive cross-peaks for intermediate correlation times

Q5. In a phase-sensitive COSY spectrum what do diagonal and off-diagonal (cross) peaks represent?

• Diagonal peaks are noise; cross-peaks are solvent signals
• Diagonal peaks correspond to the same chemical shift (auto-correlation); cross-peaks indicate scalar-coupled spins (connectivity)
• Diagonal peaks indicate spatial proximity; cross-peaks indicate through-bond J-coupling
• Both diagonal and cross-peaks indicate identical phenomena and are indistinguishable

Correct Answer: Diagonal peaks correspond to the same chemical shift (auto-correlation); cross-peaks indicate scalar-coupled spins (connectivity)

Q6. What is the main advantage of DQF-COSY (Double Quantum Filtered COSY) compared to basic COSY?

• It enhances solvent signals for easier suppression
• It reduces diagonal intensity and improves resolution of cross-peaks by filtering double-quantum coherences
• It converts through-space NOE into through-bond correlations
• It eliminates the need for t1 evolution

Correct Answer: It reduces diagonal intensity and improves resolution of cross-peaks by filtering double-quantum coherences

Q7. How does molecular tumbling (correlation time, τc) affect the sign and magnitude of NOE?

• NOE sign is always positive and independent of τc
• Fast tumbling (small molecules) typically gives positive NOE; slow tumbling (large molecules) can give negative NOE; intermediate τc can lead to near-zero NOE
• τc only affects COSY, not NOE
• Increasing τc always increases NOE magnitude without sign change

Correct Answer: Fast tumbling (small molecules) typically gives positive NOE; slow tumbling (large molecules) can give negative NOE; intermediate τc can lead to near-zero NOE

Q8. On what distance dependence does NOE intensity primarily rely when used as a distance constraint?

• Proportional to 1/r
• Proportional to 1/r2
• Proportional to 1/r3
• Proportional to 1/r6

Correct Answer: Proportional to 1/r6

Q9. Which pulse sequence configuration is characteristic of a basic homonuclear COSY experiment?

• 90° – t1 – 90° – tm – acquisition
• 180° – t1 – 90° – acquisition
• 90° – 90° with t1 evolution between the pulses followed by acquisition
• Spin-lock only during t1 followed by a single 90° pulse

Correct Answer: 90° – 90° with t1 evolution between the pulses followed by acquisition

Q10. What is “spin diffusion” in the context of NOESY and why is it undesirable?

• Accelerated T1 relaxation that increases signal-to-noise
• Indirect magnetization transfer via relay through intermediate spins during long mixing times, which produces false long-range cross-peaks
• Deliberate suppression of diagonal peaks to enhance cross-peaks
• Conversion of scalar couplings into dipolar couplings

Correct Answer: Indirect magnetization transfer via relay through intermediate spins during long mixing times, which produces false long-range cross-peaks

Q11. How does TOCSY (Total Correlation Spectroscopy) differ fundamentally from COSY and NOESY?

• TOCSY detects through-space dipolar contacts like NOESY
• TOCSY uses a prolonged spin-lock during mixing to transfer magnetization across an entire coupled spin network (through-bond isotropic mixing)
• TOCSY primarily measures relaxation times T1 and T2
• TOCSY provides only diagonal peaks with no connectivity information

Correct Answer: TOCSY uses a prolonged spin-lock during mixing to transfer magnetization across an entire coupled spin network (through-bond isotropic mixing)

Q12. In stereochemical assignments of chiral centers, how is NOESY most commonly applied?

• By measuring scalar J-couplings to determine dihedral angles directly
• By providing spatial proximity information (through-space contacts) that support relative configuration and conformation assignments
• By quantifying molecular weight through NOE intensities
• By eliminating all solvent interactions prior to structure determination

Correct Answer: By providing spatial proximity information (through-space contacts) that support relative configuration and conformation assignments

Q13. Which parameter directly links NOE buildup to internuclear distance in the initial-rate approximation?

• Scalar coupling constant J
• Cross-relaxation rate (σ), which is proportional to 1/r6
• Spin-lock field strength
• T2 relaxation time only

Correct Answer: Cross-relaxation rate (σ), which is proportional to 1/r6

Q14. Why is solvent (water) suppression important for NOESY and COSY experiments on aqueous samples?

• Water suppression increases J-coupling constants
• The large water signal can overwhelm and distort solute cross-peaks; suppression methods like presaturation or WATERGATE are required
• Water suppression converts NOE into scalar coupling
• Solvent suppression is unnecessary in two-dimensional experiments

Correct Answer: The large water signal can overwhelm and distort solute cross-peaks; suppression methods like presaturation or WATERGATE are required

Q15. What advantage does E.COSY (Exclusive COSY) provide in coupling constant measurement?

• It eliminates the need for phase cycling
• It separates multiplet components along the second dimension, allowing accurate measurement of small J-couplings
• It provides direct distance measurements via 1/r6 dependence
• It converts all cross-peaks to diagonal peaks for simplicity

Correct Answer: It separates multiplet components along the second dimension, allowing accurate measurement of small J-couplings

Q16. What are zero-quantum (ZQ) artifacts in NOESY/COSY and how are they commonly suppressed?

• ZQ artifacts are solvent peaks and are suppressed by lowering temperature
• ZQ artifacts arise from residual zero-quantum coherences causing false cross-peaks; they are suppressed by phase cycling, pulsed field gradients, or appropriate pulse timing
• ZQ artifacts are intentionally generated to enhance long-range NOEs
• ZQ artifacts are due to incomplete 90° pulses and cannot be reduced practically

Correct Answer: ZQ artifacts arise from residual zero-quantum coherences causing false cross-peaks; they are suppressed by phase cycling, pulsed field gradients, or appropriate pulse timing

Q17. What is a typical sample concentration strategy for small-molecule NOESY experiments to avoid intermolecular NOEs?

• Use extremely high concentration (>100 mM) to maximize signal
• Use moderate concentration in the low millimolar range to minimize intermolecular NOEs and aggregation
• Always use micromolar concentrations regardless of solubility
• Concentration is irrelevant for NOESY experiments

Correct Answer: Use moderate concentration in the low millimolar range to minimize intermolecular NOEs and aggregation

Q18. In a COSY spectrum, what information does an off-diagonal cross-peak between chemical shifts δA and δB provide?

• δA and δB belong to the same spin system and are scalar (J) coupled
• δA and δB are chemically equivalent and should be averaged
• δA and δB indicate solvent-exchange peaks only
• δA and δB correspond to two different molecules and cannot be related

Correct Answer: δA and δB belong to the same spin system and are scalar (J) coupled

Q19. How does typical mixing time in COSY compare with NOESY and why?

• COSY uses very long mixing times like NOESY to observe dipolar transfers
• COSY mixing is effectively very short (ms) because transfer occurs via J-coupling during the pulse sequence; NOESY requires much longer mixing times (tens to hundreds of ms) for dipolar cross-relaxation
• COSY requires no timing parameters and has infinite mixing time
• Both COSY and NOESY use identical mixing times by convention

Correct Answer: COSY mixing is effectively very short (ms) because transfer occurs via J-coupling during the pulse sequence; NOESY requires much longer mixing times (tens to hundreds of ms) for dipolar cross-relaxation

Q20. Which of the following describes a primary application of NOESY in pharmaceutical research?

• Quantifying protein molecular weight directly from chemical shifts
• Providing spatial proximity constraints to determine ligand conformations, binding modes, aggregation state and three-dimensional structures of small molecules and complexes
• Replacing mass spectrometry for purity analysis
• Measuring absolute concentrations without calibration

Correct Answer: Providing spatial proximity constraints to determine ligand conformations, binding modes, aggregation state and three-dimensional structures of small molecules and complexes

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