Fractionation and purification strategies for phytoconstituents MCQs With Answer

Introduction

Fractionation and purification of phytoconstituents are central skills for M.Pharm students specializing in phytochemistry. This blog provides a concise, exam-focused series of multiple-choice questions that explore strategic decision-making in extracting, concentrating, and purifying natural products. Topics include solvent selection and polarity, chromatographic and non-chromatographic separation methods, bioassay-guided fractionation, analytical detection techniques, sample handling for thermolabile molecules, scale-up considerations, and modern selective approaches such as molecularly imprinted polymers and counter-current chromatography. Each question targets conceptual understanding and practical choices encountered in laboratory and industry settings, helping students prepare for advanced coursework and research in natural product isolation.

Q1. Which property primarily determines the choice of solvent system for partition-based liquid–liquid extraction of plant metabolites?

• Boiling point of the solvent
• Dielectric constant and polarity of the analyte
• Color of the solvent
• Availability of glassware

Correct Answer: Dielectric constant and polarity of the analyte

Q2. In normal-phase thin-layer chromatography (silica gel), how does Rf change for a more polar phytoconstituent when using a nonpolar mobile phase?

• Rf increases because polar compounds travel faster
• Rf decreases because polar compounds are retained more strongly
• Rf remains unchanged regardless of polarity
• Rf becomes unpredictable and random

Correct Answer: Rf decreases because polar compounds are retained more strongly

Q3. What is the main advantage of bioassay-guided fractionation in natural product discovery?

• It guarantees isolation of a single pure compound in one step
• It prioritizes fractions based on biological activity to focus isolation efforts
• It reduces the need for analytical instrumentation
• It eliminates all interfering plant pigments automatically

Correct Answer: It prioritizes fractions based on biological activity to focus isolation efforts

Q4. Which detector is most suitable for detecting phytoconstituents lacking strong UV chromophores during HPLC analysis?

• Diode array detector (DAD)
• Ultraviolet (UV) detector at 254 nm
• Evaporative light scattering detector (ELSD)
• Refractive index detector (RID) for gradient elution

Correct Answer: Evaporative light scattering detector (ELSD)

Q5. What is the ideal partition coefficient (K) range for compounds during counter-current chromatography to achieve efficient separation?

• K < 0.1
• K between 0.5 and 2.0
• K > 10
• Any K value is acceptable with no impact

Correct Answer: K between 0.5 and 2.0

Q6. Which stationary phase is most appropriate for reversed-phase preparative HPLC when purifying moderately polar plant phenolics?

• Sodium chloride packed column
• Silica gel (normal phase)
• C18 (octadecyl) bonded silica
• Size-exclusion Sephadex LH-20 only

Correct Answer: C18 (octadecyl) bonded silica

Q7. Salting-out (adding saturated NaCl) during liquid–liquid extraction of aqueous plant extracts is used to:

• Increase water solubility of organics so they remain in the aqueous phase
• Increase partitioning of certain organics into the organic phase by reducing their water solubility
• Denature proteins to facilitate chromatography
• Neutralize acidic compounds to change pH

Correct Answer: Increase partitioning of certain organics into the organic phase by reducing their water solubility

Q8. Which method is most appropriate to remove a volatile organic solvent from a concentrated plant extract without overheating thermolabile compounds?

• Direct hot-plate evaporation at 120°C
• Rotary evaporation under reduced pressure with controlled bath temperature
• Air drying at room temperature only
• Microwave irradiation

Correct Answer: Rotary evaporation under reduced pressure with controlled bath temperature

Q9. Which chromatographic technique provides high selectivity for a single target phytoconstituent based on its specific binding interactions?

• Size-exclusion chromatography
• Normal-phase silica column
• Affinity chromatography using immobilized ligand or antibody
• Open-column gravity chromatography with alumina

Correct Answer: Affinity chromatography using immobilized ligand or antibody

Q10. When scaling up an extraction process from analytical to preparative scale, which parameter is most critical to reassess?

• Particle size of analytical standards only
• Solvent-to-solid ratio, mixing efficiency, and mass transfer kinetics
• Type of pen used for labeling samples
• Color of the extraction vessel

Correct Answer: Solvent-to-solid ratio, mixing efficiency, and mass transfer kinetics

Q11. Which stationary phase would you choose for fractionating high-molecular-weight tannins to separate them from small phenolic acids?

• Reverse-phase C8 column
• Size-exclusion chromatography (e.g., Sephadex LH-20)
• Ion-exchange resin for cation exchange
• Thin-layer silica with hexane as mobile phase

Correct Answer: Size-exclusion chromatography (e.g., Sephadex LH-20)

Q12. Dereplication during early stages of fractionation is primarily intended to:

• Increase the number of fractions to test
• Avoid re-isolation of known compounds by rapid identification using LC-MS/NMR databases
• Replace the need for biological assays
• Convert all compounds to volatile derivatives

Correct Answer: Avoid re-isolation of known compounds by rapid identification using LC-MS/NMR databases

Q13. Which extraction technique is considered greener and particularly useful for nonpolar phytoconstituents due to tunable solvent density and low toxicity?

• Supercritical CO2 extraction
• Petroleum ether Soxhlet with long reflux
• Soxhlet using benzene
• Direct distillation of crude plant matter

Correct Answer: Supercritical CO2 extraction

Q14. For isolating ionizable alkaloids from plant matrix, which adjustment enhances extraction into organic solvent?

• Acidify the aqueous layer to protonate alkaloids before extraction into nonpolar solvent
• Make the aqueous layer basic to convert alkaloids to free base before extraction into organic solvent
• Freeze the sample to precipitate impurities
• Always use hexane irrespective of pH

Correct Answer: Make the aqueous layer basic to convert alkaloids to free base before extraction into organic solvent

Q15. Which technique is most suitable for concentrating and purifying polar glycosides while preserving their heat-labile sugar moieties?

• Heating at high temperature for prolonged times
• Lyophilization (freeze-drying) following aqueous fractionation
• Direct crystallization from hot ethanol
• Acidic hydrolysis prior to purification

Correct Answer: Lyophilization (freeze-drying) following aqueous fractionation

Q16. What is the primary benefit of using gradient elution in reversed-phase HPLC for complex plant extracts?

• It keeps mobile phase composition constant
• It increases separation efficiency by eluting a wide polarity range of analytes in one run
• It eliminates the need for column equilibration
• It prevents detection by mass spectrometry

Correct Answer: It increases separation efficiency by eluting a wide polarity range of analytes in one run

Q17. Which approach helps reduce co-elution of similar phytoconstituents during chromatographic purification?

• Using the same solvent system at all stages
• Orthogonal purification combining two different separation principles (e.g., reverse-phase then size-exclusion)
• Reducing sample clean-up to zero
• Pooling all fractions regardless of profile

Correct Answer: Orthogonal purification combining two different separation principles (e.g., reverse-phase then size-exclusion)

Q18. Which sorbent is commonly used in solid-phase extraction (SPE) for initial cleanup of moderately polar plant metabolites before HPLC?

• C18-bonded silica cartridges
• Pure activated charcoal only
• Filter paper
• Gold-coated magnetic beads

Correct Answer: C18-bonded silica cartridges

Q19. Which factor most influences recovery losses of target phytoconstituents during multiple purification steps?

• Color of the fraction
• Adsorption to glass or silica surfaces, decomposition, and incomplete transfer between steps
• Brand of pipette tips only
• Type of personal protective equipment worn

Correct Answer: Adsorption to glass or silica surfaces, decomposition, and incomplete transfer between steps

Q20. When choosing between enrichment (concentration) and purification strategies, which statement best describes the practical rationale?

• Enrichment is always preferred because it gives 100% pure compounds
• Purification is unnecessary if enrichment achieves higher mass yield
• Start with enrichment to concentrate active fractions, then apply targeted purification to minimize loss and workload
• Purification should be performed first and enrichment later for greater efficiency

Correct Answer: Start with enrichment to concentrate active fractions, then apply targeted purification to minimize loss and workload

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