Chapter 25 – High-Performance Liquid Chromatography 1

Multiple-Choice

25-1 The Chromatographic Process

1. As particle size decreases for HPLC, the required solvent pressure

, the plate number , and the resolution
?

A) increases; increases; decreases

B) decreases; decreases; increases
C) increases; increases; increases
D) increases; decreases; decreases
E) decreases; increases; increases

Answer: C
Intermediate

Solution
The penalty for small particle size is resistance to solvent flow. The pressure required to drive
uv
solvent through a column is P=f 2 2
π r dp
One reason why small particles give better resolution is that they provide more uniform flow
through the column, thereby reducing the multiple path term, A, in the van Deemter equation. A
second reason is that the distance through which solute must diffuse in the mobile and stationary
phases is on the order of the particle size. The smaller the particles, the less distance solute must
diffuse. This effect decreases the C term in the van Deemter equation for finite equilibration
time. The optimum flow rate for small particles is faster than for large particles because solutes
diffuse through smaller distances.

2. Which of the following are characteristics of reverse-phase chromatography?

I The stationary phase is polar.

II The mobile phase is more polar than the stationary phase.
III Less polar mobile phase has a lower eluent strength.
IV More polar mobile phase has a higher eluent strength.
V The stationary phase is nonpolar.

A) I and IV
B) II only
C) I and III
D) II and IV
E) II and V

Answer: E
Intermediate
Chapter 25 – High-Performance Liquid Chromatography 2

Solution
I The stationary phase is polar.
⤷ nonpolar
III Less polar mobile phase has a lower eluent strength.
⤷ higher
IV More polar mobile phase has a higher eluent strength.
⤷ Less

3. Gradient elution is used in HPLC. How is the gradient created for a reverse-phase HPLC
experiment?

A) Increase the pH of the mobile phase over time by adding strong base.
B) Decrease the pH of the mobile phase over time by adding strong acid.
C) Increase the vapor pressure of the analytes over time by increasing the temperature.
D) Increase the polarity of the mobile phase over time by mixing with a more polar solvent.
E) Decrease the polarity of the mobile phase over time by mixing with a less polar solvent.

Answer: E
Intermediate

Solution
If one solvent does not provide sufficiently rapid elution of all components, then gradient
elution can be used. In this case, continuous change of solvent composition to increase eluent
strength. In a reversed-phase separation, eluent strength decreases as the solvent becomes more
polar.
In gradient elution, increasing amounts of solvent B are added to solvent A to create a
continuous gradient. Typically, solvent A means a water-soluble solvent, and solvent B means an
organic solvent.

4. The pressure required to push solvent through the column is dependent on several
variables such as column length, particle diameter, etc. For column pressure, which relationship
is INCORRECT?

A) Pressure is inversely proportional to column length.

B) Pressure is directly proportional to solvent viscosity.
C) Pressure is inversely proportional to particle diameter squared.
D) Pressure is inversely proportional to column radius squared.
E) Pressure is directly proportional to volume flow rate.

Answer: A
Intermediate

Solution
A) Pressure is inversely proportional to column length.
Chapter 25 – High-Performance Liquid Chromatography 3

u v nL
The pressure required to drive solvent through a column is P=f 2 2 , where u v is linear flow
π r dp
rate, n is the viscosity of the solvent, L is the length of the column, r is column radius, and d p is
the particle diameter. The factor f depends on particle shape and particle packing.

5. Which statement(s) is(are) NOT true for partition and adsorption chromatography?

I For adsorption chromatography – the lower the eluent strength of the solvent, the more
easily it displaces the solute.
II For partition chromatography – the more time the solute spends in the mobile phase, the
easier it is to elute.
III For partition chromatography – solute equilibrates between the mobile phase and the
liquid phase bonded to the solid support.
IV For adsorption chromatography – solvent molecules compete with solute molecules for
binding sites on the stationary phase.

A) IV
B) I
C) III and IV
D) II
E) I and II

Answer: B
Intermediate

Solution
I For adsorption chromatography – the lower the eluent strength of the solvent, the more
easily it displaces the solute. ⤷ higher

6. Elution with a constant composition mobile phase is:

A) constant polarity elution.

B) isopolar elution.
C) isocratic elution.
D) homogeneous elution.
E) true elution.

Answer: C
Easy

Solution
Isocratic elution is performed with a single solvent (or constant solvent mixture).
Chapter 25 – High-Performance Liquid Chromatography 4

7. Normal-phase chromatography uses a stationary phase and a

mobile phase. The more the mobile phase becomes, the stronger the
eluent strength.

A) polar; polar; polar

B) nonpolar; nonpolar; polar
C) polar; nonpolar; nonpolar
D) polar; nonpolar; polar
E) nonpolar; polar; nonpolar

Answer: D
Intermediate

Solution
Normal chromatography uses a polar stationary phase and a relatively low polarity solvent.
Therefore, as the polarity of the solvent increases, the eluent strength increases.

8. HPLC operation requires special solvents, HPLC-grade. Additionally, HPLC-grade

solvents require pretreatment before use. Which of the following is NOT a characteristic of
HPLC-grade solvents or pretreatment required before operation?

A) HPLC-grade solvent prevents degradation of costly columns with impurities.

B) HPLC-grade solvent must be sparged prior to use if the instrument is not equipped with a
degrasser.
C) HPLC-grade solvent minimizes background contamination signals in the detector.
D) HPLC-grade solvent must be filtered to remove particulate matter.
E) HPLC-grade solvent must be stored in special cabinetry to prevent solvent degradation.

Answer: E
Intermediate

Solution
E) HPLC-grade solvent must be stored in special cabinetry to prevent solvent degradation.

Very pure HPLC-grade (expensive) solvents are required to prevent degradation of costly
columns with impurities and to minimize detector background signals from contaminants.
HPLC solvents are flammable and should be stored in a secure metal cabinet when not in use.

25-2 Injection and Detection in HPLC

9. The creates gradients from up to four solvents by proportioning

liquids through a four-way valve before the piston and then pumped into the column.

A) high-pressure mixing pump

B) multiple source mixing pump
C) low-pressure mixing pump
Chapter 25 – High-Performance Liquid Chromatography 5

D) gradient mixing pump

E) variable composition mixing pump

Answer: C
Intermediate

Solution
Gradients can be delivered in two ways. In a high-pressure mixing pump each solvent is
delivered by a different pump ( solvent A from pump 1 and solvent B from pump 2). The relative
flow rates from each pump will determine the mobile phase composition.
Alternatively, in a low-pressure mixing pump, gradients made from up to four solvents are
constructed by proportioning liquids through a four-way valve at low pressure before the piston
and then pumping the mixture at high pressure into the column.

10. Which of the following is NOT true for HPLC sample injection.

A) Reproducibly load injector loop with < 0.5 or > 3 loop volumes depending on injection
technique.
B) The injection loops must be removed and cleaned after each injection.
C) Sample solvent strength should be less than or equal to eluent solvent strength.
D) Pass samples through a 0.5-m filter to remove particulate matter prior to injection.
E) Use a blunt HPLC syringe needle, not pointed, to prevent damage to the injection port.

Answer: B
Intermediate

Solution
B) The injection loops must be removed and cleaned after each injection.
The injection valve has interchangeable sample loops, each of which holds a fixed volume.

11. Which of the following is NOT a detector used with HPLC?

A) electrochemical detector
B) ultraviolet detector
C) refractive index detector
D) evaporative light-scattering detector
E) thermal conductivity detector

Answer: E
Intermediate

Solution
E) thermal conductivity detector
Thermal conductivity detector is used in gas chromatography.
Chapter 25 – High-Performance Liquid Chromatography 6

12. The responds to analytes that are significantly less volatile than
the mobile phase.

A) electrochemical detector
B) ultraviolet detector
C) refractive index detector
D) evaporative light-scattering detector
E) thermal conductivity detector

Answer: D
Intermediate

Solution
An evaporative light-scattering detector responds to any analyte that is significantly less volatile
than the mobile phase

A) electrochemical detector
An electrochemical detector responds to analytes that can be oxidized or reduced, such as
phenols, aromatic amines, peroxides, mercaptans, ketones, aldehydes, conjugated nitriles,
aromatic halogen compounds, and aromatic nitro compounds.

B) ultraviolet detector
An ultraviolet detector using a flow cell such as that is the most common HPLC detector,
because many solutes absorb ultraviolet light. Simple systems employ the intense 254-nm
emission of a mercury lamp.

C) refractive index detector

A refractive index detector responds to almost every solute, but its detection limit is about 1 000
times poorer than that of the ultraviolet detector. The deflection-type detector in Figure 25-21
has two triangular 5- to compartments through which pure solvent or eluate passes. Collimated
(parallel) visible light filtered to remove infrared radiation (which would heat the sample) passes
through the cell with pure solvent in both compartments and is directed to a photodiode array by
the deflection plate. When solute with a different refractive index enters the cell, the beam is
deflected and different pixels of the array are irradiated.

E) thermal conductivity detector

In the past, thermal conductivity detectors were most common in gas chromatography because
they are simple and universal: They respond to all analytes. Unfortunately, thermal conductivity
is not sensitive enough to detect minute quantities of analyte eluted from open tubular columns
smaller than 0.53 mm in diameter. Thermal conductivity detectors are still used for 0.53-mm
columns and for packed columns.

13. The is a sensitive, almost universal detector that responds to all

nonvolatile compounds, regardless of their structure.

A) electrochemical detector
Chapter 25 – High-Performance Liquid Chromatography 7

B) charged aerosol detector

C) refractive index detector
D) evaporative light-scattering detector
E) thermal conductivity detector

Answer: B
Intermediate

Solution
The charged aerosol detector(CAD) is a sensitive, almost universal detector that responds to all
nonvolatile compounds, regardless of their structure. For example, it is useful for measuring the
material balance in pharmaceutical preparations because the relative area of each peak in the
chromatogram is nearly equal to the relative mass of that component in the preparation.

25-3 Method Development for Isocratic Reversed-Phase Separations

14. The criteria for an adequate separation for isocratic reverse-phase HPLC include which of
the following?

I The retention factor for all peaks are in the range of 0.5 – 20.
II All peaks should be symmetric with an asymmetry factor B/A in the range of 0.9 – 15.
III For quantification, the minimum resolution between the two closest peaks should be 1.5,
though 2.0 is best to compensate for small changes in separation conditions.
IV The pressure must be less than or equal to 15 MPa to prolong instrument life.

A) I, II, III, and IV

B) I, II, and IV
C) I, II, and III
D) II, III, and IV
E) III and IV

Answer: A
Intermediate

Solution
Attributes of a good separation
• 0.5 ≤ k ≤20
• resolution ≥ 2
• operating pressure ≤ 15 MPa
• 0.9 ≤ asymmetry factor ≤ 1.5

15. During method optimization, the relative retention is adjusted by altering all of the
following, except:

A) column temperature.
B) type of organic solvent.
Chapter 25 – High-Performance Liquid Chromatography 8

C) solvent composition.
D) adjusting sample injection volume.
E) column type.

Answer: D
Intermediate

Solution
Adjustment of the relative retention may be done in a number of ways:
• Fine-tune solvent composition
• Vary column temperature
• Change the polarity of organic solvent
• Change the stationary phase

25-4 Gradient Separations

16. When deciding between an isocratic elution or a gradient elution, a scouting gradient
should be done. Which of the criteria below is INCORRECT?

A) If t/tG < 0.25, use isocratic elution.

B) If t/tG > 0.40, use gradient elution.
C) For 0.25 < t/tG < 0.40 use either an isocratic or gradient elution, the decision depending
on factors such as available equipment or complexity of the sample.
D) For t/tG < 0.25, a good starting isocratic elution solvent composition is the composition
halfway through the t of the scouting gradient.
E) For 0.25 < t/tG < 0.40, a good starting isocratic elution solvent composition is the
composition halfway through the tG of the scouting gradient.

Answer: E
Hard

Solution
E) For 0.25 < t/tG < 0.40, a good starting isocratic elution solvent composition is the
composition halfway through the tG of the scouting gradient.

If 0.25 < t/tG < 0.40, then either isocratic or gradient elution may be appropriate. Other factors
such as available equipment and complexity of the sample will influence which mode is selected.

17. A is a gradient developed to retain the resolution of a single

gradient, but reduces the analysis time.

A) broken gradient
B) segmented gradient
C) choppy gradient
D) step gradient
E) parcel gradient
Chapter 25 – High-Performance Liquid Chromatography 9

Answer: B
Easy

Solution
The segmented gradient provides an appropriate solvent composition for each region of the
chromatogram.

18. is the volume between the point at which the solvents are mixed
and the beginning of the column. Which is NOT true for dwell volume and dwell time?

A) If the dwell time is 5 minutes, a solvent change initiated at 9 minutes does not reach the
column until 14 minutes.
B) A difference in dwell volumes is the main reason why gradient separations do not always
transfer well between instruments.
C) Dwell volume is the volume between where solvents are mixed and the beginning of the
column.
D) The product of the dwell volume and the flow rate is the dwell time.
E) It is helpful to indicate the dwell volume when reporting a gradient separation.

Answer: D
Intermediate

Solution
D) The product of the dwell volume and the flow rate is the dwell time

The volume between the point at which solvents are mixed and the beginning of the column is
called the dwell volume. The dwell time is the time required for the gradient to reach the column.

Calculated

25-1 The Chromatographic Process

19. What particle diameter is needed to give 15 000 plates over a 10-cm HPLC column?
ÝÝ→

Answer: 2 m
Intermediate
Simple plug-and-calculate problem, using equation 25-1.

Solution
3000× L(cm)
N≈
d p ( μm)
3000× 10(cm) 30000
15000 ≈ → dp = = 2 μm
d p (μm) 15000
Chapter 25 – High-Performance Liquid Chromatography 10

20. What is the plate height for a 10.0-cm HPLC column packed with 3.5 m solid phase?

Answer: 1.17 x 10−3 cm or 11.7 m

Hard
Combine equation 25-1 with H = N/L to calculate the plate height. H = 3000 L2/dp.
⤷ L/N ⤷ dp/3000
Solution
3000× L(cm)
N≈
d p ( μm)
L 10 cm 10 cm
L
H=¿ = 3000 × L(cm) = 3000 ×10 (cm) = 3000 ×10 (cm) = 1.17 × 10-3
N
d p (μm) 3.5(μm) 0.00035(c m)