750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 1.** Which chromatographic parameter is defined as the ratio of the
time a solute spends on the stationary phase to the time it spends in the mobile
phase?
A) Retention time (t_R)
B) Retention factor (k)
C) Selectivity (α)
D) Column efficiency (N)
Answer: B
Explanation: The retention factor k = (t_R – t_M)/t_M, representing the relative
time a solute is retained on the column versus traveling in the mobile phase.
**Question 2.** In the Van Deemter equation, which term primarily describes
band broadening caused by differences in flow paths through the packed
particles?
A) A term (eddy diffusion)
B) B term (longitudinal diffusion)
C) C term (mass transfer)
D) D term (instrumental noise)
Answer: A
Explanation: The A term reflects eddy diffusion, arising from multiple flow paths
of different lengths through the packed bed, leading to peak widening.
, 750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 3.** What is the main advantage of using superficially porous
(core‑shell) particles in UPLC columns compared with fully porous sub‑2 µm
particles?
A) Higher back‑pressure at the same flow rate
B) Faster mass‑transfer kinetics with lower plate height
C) Greater particle surface area for ion‑exchange
D) Improved compatibility with normal‑phase separations
Answer: B
Explanation: Core‑shell particles have a thin porous shell surrounding a solid core,
reducing diffusion distances and thus providing faster mass‑transfer and lower
plate heights at comparable pressures.
**Question 4.** Which mobile‑phase additive is most suitable for electrospray
ionization mass spectrometry (ESI‑MS) when analysing basic compounds?
A) Trifluoroacetic acid (TFA)
B) Formic acid
C) Ammonium acetate
D) Phosphoric acid
Answer: C
Explanation: Ammonium acetate is a volatile buffer that maintains pH without
suppressing ionization, making it ideal for ESI‑MS; TFA can suppress ionization of
basic analytes.
, 750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 5.** When preparing a gradient method for a reversed‑phase UPLC
system, which parameter must be adjusted to maintain equivalent gradient
steepness after scaling from an HPLC method?
A) Gradient dwell volume
B) Column temperature
C. Detector wavelength
D) Injection volume
Answer: A
Explanation: Gradient dwell volume (the volume between the mixing point and
the column inlet) influences the effective gradient shape; scaling methods must
account for differences in dwell volume between HPLC and UPLC.
**Question 6.** A chromatogram shows a systematic increase in retention time
after every injection. Which of the following is the most likely cause?
A) Column temperature drift upward
B) Mobile‑phase composition becoming more aqueous
C) Pump pressure decreasing gradually
D) Detector lamp aging
Answer: B
Explanation: An increasingly aqueous mobile phase reduces elution strength,
causing later retention. Temperature drift would typically cause earlier retention if
temperature rises.
, 750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 7.** Which of the following pump designs is least prone to pulsation
in gradient delivery?
A) Single‑piston reciprocating pump
B) Dual‑piston reciprocating pump with check valves
C) Triple‑piston pump with low‑pressure mixing
D) Diaphragm pump with variable flow control
Answer: C
Explanation: Triple‑piston pumps allow high‑pressure mixing of solvents,
minimizing pulsation and providing smoother gradients compared with
single‑piston designs.
**Question 8.** In a six‑port injection valve, what is the purpose of the “sample
loop” when operating in full‑loop mode?
A) To trap air bubbles before they enter the column
B) To hold a precise volume of sample that is completely transferred to the
column on each injection
C) To mix the sample with the mobile phase before injection
D) To clean the needle after each injection
Answer: B
Explanation: In full‑loop mode, the loop is filled with the exact injection volume;
the entire contents are transferred to the column each time, ensuring
reproducible injection amounts.
Troubleshooting Practice Exam
**Question 1.** Which chromatographic parameter is defined as the ratio of the
time a solute spends on the stationary phase to the time it spends in the mobile
phase?
A) Retention time (t_R)
B) Retention factor (k)
C) Selectivity (α)
D) Column efficiency (N)
Answer: B
Explanation: The retention factor k = (t_R – t_M)/t_M, representing the relative
time a solute is retained on the column versus traveling in the mobile phase.
**Question 2.** In the Van Deemter equation, which term primarily describes
band broadening caused by differences in flow paths through the packed
particles?
A) A term (eddy diffusion)
B) B term (longitudinal diffusion)
C) C term (mass transfer)
D) D term (instrumental noise)
Answer: A
Explanation: The A term reflects eddy diffusion, arising from multiple flow paths
of different lengths through the packed bed, leading to peak widening.
, 750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 3.** What is the main advantage of using superficially porous
(core‑shell) particles in UPLC columns compared with fully porous sub‑2 µm
particles?
A) Higher back‑pressure at the same flow rate
B) Faster mass‑transfer kinetics with lower plate height
C) Greater particle surface area for ion‑exchange
D) Improved compatibility with normal‑phase separations
Answer: B
Explanation: Core‑shell particles have a thin porous shell surrounding a solid core,
reducing diffusion distances and thus providing faster mass‑transfer and lower
plate heights at comparable pressures.
**Question 4.** Which mobile‑phase additive is most suitable for electrospray
ionization mass spectrometry (ESI‑MS) when analysing basic compounds?
A) Trifluoroacetic acid (TFA)
B) Formic acid
C) Ammonium acetate
D) Phosphoric acid
Answer: C
Explanation: Ammonium acetate is a volatile buffer that maintains pH without
suppressing ionization, making it ideal for ESI‑MS; TFA can suppress ionization of
basic analytes.
, 750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 5.** When preparing a gradient method for a reversed‑phase UPLC
system, which parameter must be adjusted to maintain equivalent gradient
steepness after scaling from an HPLC method?
A) Gradient dwell volume
B) Column temperature
C. Detector wavelength
D) Injection volume
Answer: A
Explanation: Gradient dwell volume (the volume between the mixing point and
the column inlet) influences the effective gradient shape; scaling methods must
account for differences in dwell volume between HPLC and UPLC.
**Question 6.** A chromatogram shows a systematic increase in retention time
after every injection. Which of the following is the most likely cause?
A) Column temperature drift upward
B) Mobile‑phase composition becoming more aqueous
C) Pump pressure decreasing gradually
D) Detector lamp aging
Answer: B
Explanation: An increasingly aqueous mobile phase reduces elution strength,
causing later retention. Temperature drift would typically cause earlier retention if
temperature rises.
, 750000907 HPLC and UPLC Best Practices and
Troubleshooting Practice Exam
**Question 7.** Which of the following pump designs is least prone to pulsation
in gradient delivery?
A) Single‑piston reciprocating pump
B) Dual‑piston reciprocating pump with check valves
C) Triple‑piston pump with low‑pressure mixing
D) Diaphragm pump with variable flow control
Answer: C
Explanation: Triple‑piston pumps allow high‑pressure mixing of solvents,
minimizing pulsation and providing smoother gradients compared with
single‑piston designs.
**Question 8.** In a six‑port injection valve, what is the purpose of the “sample
loop” when operating in full‑loop mode?
A) To trap air bubbles before they enter the column
B) To hold a precise volume of sample that is completely transferred to the
column on each injection
C) To mix the sample with the mobile phase before injection
D) To clean the needle after each injection
Answer: B
Explanation: In full‑loop mode, the loop is filled with the exact injection volume;
the entire contents are transferred to the column each time, ensuring
reproducible injection amounts.
