Ion exchange chromatography (IEC) is a fundamental separation technique in pharmaceutical analysis, widely used for drug purification, protein separation, and formulation development. B. Pharm students should learn about ion-exchange resins, strong and weak cation and anion exchangers, buffer selection, pH effects, selectivity, capacity, and elution strategies to apply IEC effectively in quality control, impurity removal, salt form optimization, and biotherapeutic purification. Practical considerations include column packing, regeneration, scale-up, dynamic binding capacity, and troubleshooting issues such as peak tailing or resin fouling. This focused overview links theory to common pharmaceutical applications and laboratory practice. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the primary mechanism by which ion exchange chromatography separates analytes?
- Size exclusion through pores
- Hydrophobic interactions with stationary phase
- Electrostatic interactions between charged analytes and ion-exchange resin
- Partitioning between two immiscible solvents
Correct Answer: Electrostatic interactions between charged analytes and ion-exchange resin
Q2. Which resin would you choose to retain positively charged drug molecules at neutral pH?
- Strong anion exchanger (SAX)
- Weak anion exchanger (WAX)
- Strong cation exchanger (SCX)
- Size-exclusion resin
Correct Answer: Strong cation exchanger (SCX)
Q3. Which parameter most directly affects ionization state of an analyte and therefore its binding to the resin?
- Column length
- pH of the buffer
- Column temperature
- Detector sensitivity
Correct Answer: pH of the buffer
Q4. In pharmaceutical production, ion exchange chromatography is commonly used for which application?
- Determining melting point of excipients
- Purifying protein therapeutics and removing charged impurities
- Measuring tablet hardness
- Solubility testing
Correct Answer: Purifying protein therapeutics and removing charged impurities
Q5. A weak cation exchanger differs from a strong cation exchanger mainly in:
- Particle size of the resin
- pH-dependent ionization of functional groups
- Ability to separate neutral molecules
- Requirement for organic solvents
Correct Answer: pH-dependent ionization of functional groups
Q6. What is a typical elution strategy to displace strongly bound ions from an ion-exchange column?
- Lower buffer temperature only
- Increase ionic strength or change pH of the eluent
- Decrease detector wavelength
- Reduce column diameter
Correct Answer: Increase ionic strength or change pH of the eluent
Q7. Which salt is commonly used in gradients to elute analytes from ion exchange columns?
- Sodium chloride (NaCl)
- Magnesium sulfate
- Potassium permanganate
- Sodium dodecyl sulfate (SDS)
Correct Answer: Sodium chloride (NaCl)
Q8. What is the effect of increasing ionic strength of the mobile phase on analyte retention in IEC?
- Retention increases due to stronger binding
- Retention decreases as competing ions displace analytes
- No effect on retention
- Column pressure increases dramatically
Correct Answer: Retention decreases as competing ions displace analytes
Q9. For protein purification, why is ion exchange chromatography often chosen?
- It separates solely based on molecular weight
- It exploits differences in protein surface charge and pI for high resolution
- It requires no buffer optimization
- It uses organic solvents that denature proteins
Correct Answer: It exploits differences in protein surface charge and pI for high resolution
Q10. What does the term “dynamic binding capacity” refer to in preparative IEC?
- Total volume of the column
- Maximum analyte mass bound under flow before breakthrough
- Detector linear range
- Resin shelf life
Correct Answer: Maximum analyte mass bound under flow before breakthrough
Q11. Which of the following is a sign of column overloading in ion exchange chromatography?
- Sharper, symmetric peaks
- Peak broadening and tailing
- Increased baseline noise only
- No change in chromatogram
Correct Answer: Peak broadening and tailing
Q12. Which troubleshooting step helps reduce peak tailing caused by heterogeneous packing?
- Use a lower ionic strength buffer
- Repack the column with uniformly sized resin beads
- Increase detector gain
- Use a longer column without repacking
Correct Answer: Repack the column with uniformly sized resin beads
Q13. How does pH gradient elution differ from salt gradient elution in IEC?
- pH gradient changes the analyte charge state; salt gradient increases competition by ions
- Salt gradients change analyte charge state; pH gradients increase ionic strength
- Both are identical in mechanism
- pH gradient is only for hydrophobic compounds
Correct Answer: pH gradient changes the analyte charge state; salt gradient increases competition by ions
Q14. Which analytical application uses ion exchange to determine the counterion purity of a drug salt?
- Thermogravimetric analysis
- Ion chromatography for counterion analysis
- X-ray crystallography
- Polarimetry
Correct Answer: Ion chromatography for counterion analysis
Q15. What is the role of buffer capacity in ion exchange chromatography?
- Buffers alter column porosity
- High buffer capacity resists pH changes during sample loading and elution
- Buffers always decrease selectivity
- Buffer capacity controls detector sensitivity
Correct Answer: High buffer capacity resists pH changes during sample loading and elution
Q16. Which factor primarily determines selectivity of an ion exchange separation?
- Particle color
- Relative affinity (selectivity coefficient) of resin for different ions
- Detector brand
- Column storage temperature
Correct Answer: Relative affinity (selectivity coefficient) of resin for different ions
Q17. In ion exchange of small molecule drugs, why might one convert a drug to a specific salt form?
- To increase volatility for GC analysis
- To optimize solubility, stability, bioavailability, or chromatographic behavior
- To decrease molecular weight
- To change stereochemistry
Correct Answer: To optimize solubility, stability, bioavailability, or chromatographic behavior
Q18. Which cleaning solution is commonly used to regenerate a cation exchange resin?
- Strong base like NaOH only
- High concentration of the counterion, e.g., 1 M NaCl, or acid wash depending on resin
- Pure organic solvent
- Distilled water only
Correct Answer: High concentration of the counterion, e.g., 1 M NaCl, or acid wash depending on resin
Q19. Why is conductivity detection commonly used with ion exchange chromatography?
- It is insensitive to ions
- It directly measures ionic species eluting from the column
- It provides molecular weight information
- It detects only neutral compounds
Correct Answer: It directly measures ionic species eluting from the column
Q20. Which statement about weak ion exchangers is true?
- They have fixed charge independent of pH
- Their charge varies with pH, allowing selective binding/release around pKa
- They cannot be used for proteins
- They are always superior to strong exchangers
Correct Answer: Their charge varies with pH, allowing selective binding/release around pKa
Q21. What is a common reason to use a desalting step with ion exchange chromatography in biopharmaceuticals?
- To increase ionic strength before storage
- To remove salts and exchange buffer for downstream formulation
- To add preservatives to the product
- To decrease product purity
Correct Answer: To remove salts and exchange buffer for downstream formulation
Q22. Which of the following best describes the elution order in cation exchange chromatography for mono-, di-, and trivalent cations under similar conditions?
- Trivalent elute first, then divalent, then monovalent
- Monovalent elute first, then divalent, then trivalent
- All elute simultaneously
- Order depends only on molecular weight
Correct Answer: Monovalent elute first, then divalent, then trivalent
Q23. What is the effect of increasing flow rate on resolution in IEC, assuming all else constant?
- Resolution generally improves with very high flow rates
- Resolution generally decreases due to reduced interaction time
- Flow rate has no effect on resolution
- Resolution becomes infinite
Correct Answer: Resolution generally decreases due to reduced interaction time
Q24. Which pharmaceutical impurity removal task is well-suited to ion exchange chromatography?
- Removal of neutral organic solvents
- Separation of charged degradation products from active pharmaceutical ingredients
- Elimination of uncharged polymeric excipients
- Lyophilization of dosage forms
Correct Answer: Separation of charged degradation products from active pharmaceutical ingredients
Q25. When designing an IEC method for a protein with pI 6.5, at pH 8.0 the protein will:
- Carry a net positive charge and bind to cation exchangers
- Be neutral and not bind to any resin
- Carry a net negative charge and bind to anion exchangers
- Denature immediately regardless of resin
Correct Answer: Carry a net negative charge and bind to anion exchangers
Q26. Which factor can lead to loss of resin capacity over repeated use?
- Proper regeneration after each run
- Fouling by hydrophobic contaminants and irreversible binding
- Using appropriate buffer ionic strength
- Storing resin in recommended preservative
Correct Answer: Fouling by hydrophobic contaminants and irreversible binding
Q27. In ion exchange HPLC for small molecules, what is a reason to use a volatile buffer (e.g., ammonium acetate)?
- To prevent ionization of analytes
- Compatibility with mass spectrometry detection
- Volatile buffers increase resin strength
- They eliminate the need for column equilibration
Correct Answer: Compatibility with mass spectrometry detection
Q28. What is ion-exchange capacity of a resin typically expressed as?
- mL per minute
- Milliequivalents per gram or per milliliter of resin
- Wavelength in nm
- Column length in cm
Correct Answer: Milliequivalents per gram or per milliliter of resin
Q29. Which operational practice improves reproducibility of IEC runs?
- Skipping column equilibration between runs
- Consistent buffer preparation, column equilibration, and controlled flow rates
- Varying sample injection volume randomly
- Changing resin brand frequently
Correct Answer: Consistent buffer preparation, column equilibration, and controlled flow rates
Q30. What distinguishes ion-pair chromatography from classical ion exchange chromatography?
- Ion-pair uses a charged additive in mobile phase to form neutral pairs with analytes, enabling reversed-phase separation
- Ion-pair uses only ion-exchange resins
- Ion-pair requires paper as stationary phase
- They are identical in mechanism and application
Correct Answer: Ion-pair uses a charged additive in mobile phase to form neutral pairs with analytes, enabling reversed-phase separation
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
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