Ultra and nano-liquid chromatography MCQs With Answer

Introduction

This quiz collection on Ultra and Nano-Liquid Chromatography is designed for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). It focuses on principles, instrumentation, column technology, method transfer, and LC–MS interfacing specific to UHPLC and nano‑LC. Questions emphasize practical aspects such as particle size effects, high-pressure operation, nanospray ionization, extra‑column effects, trapping strategies, and troubleshooting of low‑flow systems. Each MCQ challenges you to apply theoretical knowledge to routine and research problems encountered in pharmaceutical analysis, proteomics, and trace‑level quantitation. Use these questions to test and deepen your understanding before exams or lab work.

Q1. What is the primary chromatographic change achieved by UHPLC compared to conventional HPLC?

• Use of larger particle sizes to reduce backpressure
• Use of sub‑2 µm stationary phase particles to increase efficiency at higher pressures
• Replacement of liquid mobile phase with supercritical fluids
• Exclusive use of very wide bore columns for larger sample loads

Correct Answer: Use of sub‑2 µm stationary phase particles to increase efficiency at higher pressures

Q2. What is the main analytical advantage of nano‑LC when coupled to electrospray mass spectrometry?

• Higher flow rates leading to shorter analysis time
• Reduced solvent consumption without sensitivity gain
• Improved ionization efficiency and sensitivity due to low flow (nL/min) electrospray
• Ability to use larger injection volumes for complex samples

Correct Answer: Improved ionization efficiency and sensitivity due to low flow (nL/min) electrospray

Q3. Which inner diameter is most commonly used for analytical nano‑LC columns in proteomics?

• 4.6 mm
• 1.0 mm
• 75 µm
• 2.1 mm

Correct Answer: 75 µm

Q4. Decreasing particle size in a column most directly reduces which contribution to the Van Deemter equation?

• The A term (eddy diffusion) and reduces mass transfer distances (C term) improving H
• The B term only (longitudinal diffusion)
• Only the mobile phase viscosity
• Only the column dead volume

Correct Answer: The A term (eddy diffusion) and reduces mass transfer distances (C term) improving H

Q5. What is the principal technical limitation that must be addressed when converting methods to UHPLC?

• The need for much higher sample injection volumes
• The requirement for system components rated for very high operating pressures and control of frictional heating
• The incompatibility with gradient elution
• The impossibility of using reversed‑phase stationary phases

Correct Answer: The requirement for system components rated for very high operating pressures and control of frictional heating

Q6. What is the typical flow rate range used in nano‑LC for on‑line LC‑MS analyses?

• 1–5 mL/min
• 50–500 nL/min
• 0.5–2.0 mL/min
• 10–20 µL/min

Correct Answer: 50–500 nL/min

Q7. What is the primary purpose of a trap (precolumn) used in nano‑LC workflows?

• To increase column backpressure
• To preconcentrate analytes and perform on‑line desalting before the analytical nanocolumn
• To heat the mobile phase before ionization
• To split the flow to multiple detectors

Correct Answer: To preconcentrate analytes and perform on‑line desalting before the analytical nanocolumn

Q8. Reducing column inner diameter from 4.6 mm to 75 µm while maintaining similar stationary phase increases MS sensitivity primarily because:

• The column holds more sample mass
• Lower flow rates produce more efficient electrospray ionization and higher ion transfer efficiency
• Retention times become much longer
• Mass spectrometers are calibrated for small column diameters

Correct Answer: Lower flow rates produce more efficient electrospray ionization and higher ion transfer efficiency

Q9. In a nano‑ESI setup the function of the emitter tip is to:

• Act as the primary detector for absorbance
• Generate a stable Taylor cone and produce charged droplets for efficient ionization
• Increase the backpressure to extend column life
• Deliver sheath gas into the column

Correct Answer: Generate a stable Taylor cone and produce charged droplets for efficient ionization

Q10. Which factor is most responsible for band broadening in practical nano‑LC systems despite high column efficiency?

• Excessively small particle size in the column
• Extra‑column dead volume from fittings, connectors, and the injector
• The use of gradient elution
• High sample concentrations only

Correct Answer: Extra‑column dead volume from fittings, connectors, and the injector

Q11. For shotgun proteomics using nano‑LC, which detector/interface is most commonly used?

• Refractive index detector (RID)
• Evaporative light scattering detector (ELSD)
• Electrospray ionization tandem mass spectrometry (ESI‑MS/MS)
• Fluorescence detector only

Correct Answer: Electrospray ionization tandem mass spectrometry (ESI‑MS/MS)

Q12. Why is dwell volume (gradient delay volume) critical in UHPLC method transfer and performance?

• It determines the maximum detector wavelength
• It affects gradient delay, reproducibility of retention times, and peak shape for fast gradients
• It controls column temperature directly
• It only matters for isocratic separations

Correct Answer: It affects gradient delay, reproducibility of retention times, and peak shape for fast gradients

Q13. When scaling flow to maintain linear velocity from a 4.6 mm ID column to a 2.1 mm ID column, flow rate should be changed by which factor?

• Multiply by (4.6/2.1)
• Multiply by (2.1/4.6)
• Multiply by (2.1/4.6)^2 (~0.21 of original flow)
• Keep flow rate unchanged

Correct Answer: Multiply by (2.1/4.6)^2 (~0.21 of original flow)

Q14. To minimize clogging and preserve column life in nano‑LC, best practice is to:

• Use high dead‑volume unions to trap particles
• Employ in‑line filters, low dead‑volume fittings, and guard/trap columns with suitable pore size
• Use only glass capillaries with no fittings
• Increase flow rate to flush particles through the system

Correct Answer: Employ in‑line filters, low dead‑volume fittings, and guard/trap columns with suitable pore size

Q15. What is the effect of frictional heating in narrow UHPLC columns operated at very high linear velocities?

• No measurable effect on chromatography
• Uniform cooling that increases viscosity uniformly
• Axial temperature gradients that change retention, reduce efficiency, and may distort peaks
• Only affects detector response but not retention

Correct Answer: Axial temperature gradients that change retention, reduce efficiency, and may distort peaks

Q16. Peak capacity in the context of gradient separations is best described as:

• The number of theoretical plates per meter of column
• The maximum number of components that can be resolved in a given gradient window; UHPLC and nano‑LC typically increase peak capacity by improving efficiency and reducing peak width
• The total solvent consumption per run
• The detector noise level divided by peak height

Correct Answer: The maximum number of components that can be resolved in a given gradient window; UHPLC and nano‑LC typically increase peak capacity by improving efficiency and reducing peak width

Q17. For a 75 µm ID analytical nano‑column, a recommended typical injection volume to avoid column overload is:

• 10–100 nL
• 50–200 µL
• 1–5 µL
• 1–2 mL

Correct Answer: 10–100 nL

Q18. Which system suitability parameter is most sensitive to extra‑column volume in a UHPLC/nano‑LC system?

• Detector wavelength accuracy
• Peak width and column efficiency (theoretical plates)
• Retention factor of non‑retained compounds
• Mobile phase pH

Correct Answer: Peak width and column efficiency (theoretical plates)

Q19. Which mobile phase additive is generally preferred for nano‑LC‑ESI‑MS to maximize peptide ionization while maintaining chromatographic performance?

• Trifluoroacetic acid (0.1%)
• Formic acid (0.1%)
• Sodium phosphate buffer
• High concentration ammonium chloride

Correct Answer: Formic acid (0.1%)

Q20. A common operational challenge unique to nano‑LC compared with analytical HPLC is:

• Inability to perform gradient separations
• Increased frequency of emitter/column clogging and spray instability due to small orifices and low flow
• Lack of compatibility with MS detection
• Excessive solvent consumption

Correct Answer: Increased frequency of emitter/column clogging and spray instability due to small orifices and low flow

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