Principle and procedure of Solid Phase Extraction MCQs With Answer

Introduction: Solid Phase Extraction (SPE) is a powerful sample-preparation technique widely used in pharmaceutical analysis to isolate, concentrate, and purify analytes from complex matrices. This introduction covers the principle — selective adsorption and desorption on sorbents (C18, HLB, ion-exchange, mixed-mode) — and the stepwise procedure: conditioning, sample loading, washing, drying, and elution. Key factors such as sorbent selection, pH control, solvent strength, flow rate, breakthrough volume, and cartridge capacity affect recovery and selectivity. Understanding SPE troubleshooting, automation (on-line SPE), and compatibility with LC-MS ensures reliable method development and validation for B.Pharm students. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the basic principle of Solid Phase Extraction (SPE)?

• Selective adsorption and desorption of analytes on a sorbent
• Distillation of volatile analytes from a sample
• Chemical reaction converting analytes to gases
• Direct injection of raw sample into the detector

Correct Answer: Selective adsorption and desorption of analytes on a sorbent

Q2. Which sequence correctly describes the typical SPE procedure?

• Conditioning → Sample loading → Washing → Elution
• Elution → Washing → Conditioning → Sample loading
• Washing → Conditioning → Elution → Sample loading
• Sample loading → Conditioning → Elution → Washing

Correct Answer: Conditioning → Sample loading → Washing → Elution

Q3. What is the purpose of the conditioning step in SPE?

• To dry the sorbent completely before use
• To activate, wet and equilibrate the sorbent with an appropriate solvent
• To elute analytes from the sorbent
• To neutralize the sample matrix

Correct Answer: To activate, wet and equilibrate the sorbent with an appropriate solvent

Q4. Which sorbent is most suitable for retaining nonpolar (hydrophobic) analytes in reversed-phase SPE?

• Silica gel without modification
• C18 (octadecyl) bonded silica
• Strong cation exchange resin
• Hydrophilic-lipophilic balanced (HLB) polymer

Correct Answer: C18 (octadecyl) bonded silica

Q5. Ion-exchange sorbents in SPE retain analytes primarily by which mechanism?

• Hydrophobic interactions only
• Ionic (electrostatic) interactions between charged analytes and charged sites on the sorbent
• Size exclusion based on molecular weight
• Covalent bonding to the sorbent

Correct Answer: Ionic (electrostatic) interactions between charged analytes and charged sites on the sorbent

Q6. What does the term “breakthrough volume” refer to in SPE?

• The volume of eluent required to wash the sorbent
• The volume at which the analyte begins to appear in the column effluent during sample loading
• The total capacity of solvent a cartridge can hold
• The maximum volume of eluent collected after elution

Correct Answer: The volume at which the analyte begins to appear in the column effluent during sample loading

Q7. For a weak acid analyte, which pH adjustment will generally increase retention on a reversed-phase sorbent?

• Adjust pH to be well above the analyte pKa
• Adjust pH to be well below the analyte pKa (suppress ionization)
• Adjust pH to equal the pKa exactly
• pH has no effect on retention of weak acids

Correct Answer: Adjust pH to be well below the analyte pKa (suppress ionization)

Q8. Which elution solvent is typically used to remove strongly retained analytes from a reversed-phase SPE cartridge?

• Water only
• High percentage organic solvent such as methanol or acetonitrile
• Dilute buffer of the same polarity as water
• Nonpolar gas

Correct Answer: High percentage organic solvent such as methanol or acetonitrile

Q9. What is the main aim of the wash step during SPE?

• To elute the target analyte completely
• To remove matrix interferences while retaining the analyte on the sorbent
• To dry the cartridge before elution
• To change the sorbent chemically

Correct Answer: To remove matrix interferences while retaining the analyte on the sorbent

Q10. What is a common sign that an SPE cartridge has been overloaded?

• Enhanced analyte retention and increased recovery
• Appearance of analyte in the load effluent and reduced overall recovery
• Complete exclusion of matrix components
• Instant dryness of the sorbent

Correct Answer: Appearance of analyte in the load effluent and reduced overall recovery

Q11. Compared to liquid-liquid extraction (LLE), what is a primary advantage of SPE?

• SPE always gives 100% recovery
• SPE uses less organic solvent and offers better reproducibility and automation potential
• SPE is always faster regardless of sample type
• SPE does not require any method development

Correct Answer: SPE uses less organic solvent and offers better reproducibility and automation potential

Q12. What does HLB sorbent stand for and why is it useful?

• High-Lipid Binder; used for fats only
• Hydrophilic-Lipophilic Balanced; useful for a wide polarity range of analytes
• Highly Loaded Bed; indicates very high capacity
• Hydrophilic Low Binding; used for proteins

Correct Answer: Hydrophilic-Lipophilic Balanced; useful for a wide polarity range of analytes

Q13. Why is drying the sorbent (e.g., with air or vacuum) often performed before elution?

• To oxidize the analyte
• To remove residual aqueous solvent that would dilute or reduce elution efficiency
• To increase cartridge backpressure
• To change sorbent chemistry from hydrophobic to hydrophilic

Correct Answer: To remove residual aqueous solvent that would dilute or reduce elution efficiency

Q14. How does Solid Phase Microextraction (SPME) differ fundamentally from cartridge SPE?

• SPME uses a coated fiber and is solvent-free, sampling by direct partitioning onto the coating
• SPME uses larger cartridges with higher sorbent mass
• SPME requires large volumes of organic solvent for elution
• SPME is identical to cartridge SPE but with different name

Correct Answer: SPME uses a coated fiber and is solvent-free, sampling by direct partitioning onto the coating

Q15. What is an advantage of on-line SPE coupled to LC-MS?

• It requires manual transfer of samples between instruments
• It allows direct automated sample cleanup and reduces human error and sample handling time
• It eliminates the need for chromatographic separation
• It increases solvent consumption dramatically

Correct Answer: It allows direct automated sample cleanup and reduces human error and sample handling time

Q16. A sudden increase in system backpressure during SPE is most likely caused by which problem?

• Excessive sorbent capacity
• Particulate clogging of the sorbent bed or use of too fine particle size
• Using too much organic solvent during elution
• High analyte recovery

Correct Answer: Particulate clogging of the sorbent bed or use of too fine particle size

Q17. How should the flow rate be managed during sample loading to optimize analyte retention?

• Use the fastest possible flow to speed up the process
• Use a slow flow rate to maximize contact time between analyte and sorbent
• Flow rate has no impact on retention
• Alternate high and low flow rapidly during loading

Correct Answer: Use a slow flow rate to maximize contact time between analyte and sorbent

Q18. Which sequence of conditioning solvents is commonly used for reversed-phase C18 cartridges before loading an aqueous sample?

• Water → Hexane
• Methanol (or ACN) → Water or buffer
• Strong base → Strong acid
• Dry air only

Correct Answer: Methanol (or ACN) → Water or buffer

Q19. How is the sorbent capacity usually defined for SPE cartridges?

• The amount of solvent required to elute an analyte
• The maximum amount of analyte the sorbent can retain before breakthrough occurs
• The physical volume of the cartridge housing
• The total sample volume one can process regardless of analyte amount

Correct Answer: The maximum amount of analyte the sorbent can retain before breakthrough occurs

Q20. For eluting ionically bound analytes from an ion-exchange sorbent, which approach is most effective?

• Use a nonpolar solvent like hexane
• Change pH or increase ionic strength to disrupt ionic interactions
• Reduce temperature to freeze the analyte
• Use a very dilute aqueous solution only

Correct Answer: Change pH or increase ionic strength to disrupt ionic interactions

Q21. In LC-MS analysis after SPE, what does the term “matrix effect” usually refer to?

• The physical clogging of the LC column by particulates
• Ion suppression or enhancement caused by co-eluting matrix components affecting MS response
• A change in cartridge color
• An increase in cartridge capacity over time

Correct Answer: Ion suppression or enhancement caused by co-eluting matrix components affecting MS response

Q22. Why is buffering or pH adjustment of the sample often necessary before SPE?

• To change the color of the sample for visualization
• To adjust analyte ionization state and ionic strength, optimizing retention and selectivity
• To evaporate the sample faster
• pH adjustment is never necessary for SPE

Correct Answer: To adjust analyte ionization state and ionic strength, optimizing retention and selectivity

Q23. If a polar analyte shows poor retention on a C18 cartridge, which alternative sorbent strategy is appropriate?

• Use a polar sorbent such as HILIC or a hydrophilic-lipophilic balanced (HLB) cartridge
• Increase the C18 chain length to C30
• Replace with a nonporous glass bead cartridge
• Only increase sample temperature during loading

Correct Answer: Use a polar sorbent such as HILIC or a hydrophilic-lipophilic balanced (HLB) cartridge

Q24. How is percent recovery commonly calculated after SPE cleanup?

• (Amount in original sample / Amount recovered) × 100
• (Amount recovered / Amount spiked) × 100
• (Blank signal / Sample signal) × 100
• (Elapsed time / Total time) × 100

Correct Answer: (Amount recovered / Amount spiked) × 100

Q25. Compared to cartridges, what is a typical advantage of SPE disk formats?

• Disks always provide higher selectivity
• Disks allow higher flow rates and faster processing for large-volume samples
• Disks are never compatible with automated systems
• Disks eliminate the need for conditioning

Correct Answer: Disks allow higher flow rates and faster processing for large-volume samples

Q26. Which conditioning practice helps prevent analyte breakthrough when loading samples with substantial organic content?

• Conditioning with a solvent that matches the loading solvent composition to ensure proper sorbent wettability
• Conditioning with pure hexane only
• Skipping conditioning entirely to save time
• Conditioning with strong acid only

Correct Answer: Conditioning with a solvent that matches the loading solvent composition to ensure proper sorbent wettability

Q27. Why is evaporation and reconstitution often performed after SPE elution?

• To degrade the analyte for analysis
• To concentrate the analyte and to change the solvent to one compatible with the analytical method
• To increase cartridge capacity for the next sample
• To neutralize the sorbent chemically

Correct Answer: To concentrate the analyte and to change the solvent to one compatible with the analytical method

Q28. Low analyte recovery after SPE may be caused by which of the following?

• Incomplete elution or strong irreversible binding to the sorbent
• Excessive evaporation of the eluent while collecting it
• Using the correct pH for sample loading
• Proper sorbent conditioning

Correct Answer: Incomplete elution or strong irreversible binding to the sorbent

Q29. How is selectivity in an SPE method primarily achieved?

• By randomly choosing solvents
• By matching sorbent chemistry and controlling sample pH and solvent strength
• By increasing sample temperature indiscriminately
• By using the largest possible cartridge

Correct Answer: By matching sorbent chemistry and controlling sample pH and solvent strength

Q30. What is meant by a “mixed-mode” SPE sorbent?

• A sorbent that mixes sample and eluent together
• A sorbent combining more than one retention mechanism, e.g., reversed-phase and ion-exchange
• A sorbent that randomly changes chemistry during use
• A sorbent that is only used for gas samples

Correct Answer: A sorbent combining more than one retention mechanism, e.g., reversed-phase and ion-exchange

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