Chromatography 2

Question 1

What is chromatography?

Answer 1

A technique to separate, identify, and quantify components in a mixture by passing them through a stationary phase.

Question 2

What are the two main outputs of chromatography?

Answer 2

Qualitative analysis: Identifies the components (e.g., retention time).
Quantitative analysis: Measures the concentration (e.g., peak area).

Question 3

What are the six key components of an HPLC system?

Answer 3

Mobile phase: Solvent carrying analytes.
Pump: Ensures precise flow of the mobile phase.
Injector: Introduces the sample into the system.
Column: Stationary phase where separation occurs.
Detector: Detects analytes in the mobile phase.
Recorder (chromatogram): Translates signals into a graphical output.

Question 4

What is the role of the guard column in HPLC?

Answer 4

It removes sample components that may foul the analytical column, prolonging its lifespan.

Question 5

What is retention time (
𝑡𝑅 ) in HPLC?

Answer 5

The time it takes for an analyte to travel from the injector to the detector.

Question 6

What is dead time (
𝑡0 ) in HPLC?

Answer 6

The time required for an inert compound to pass through the column without interacting with the stationary phase.

Question 7

How is resolution (
𝑅𝑠 ) calculated in HPLC?

Answer 7

Good separation requires
𝑅𝑠 ≥1.5.

Question 8

What are the two types of column packing?

Answer 8

Pellicular particles: Porous stationary phase on a solid core; used in guard columns.
Porous particles: Silica or polymer particles; used in analytical columns.

Question 9

What is the stationary phase in reverse-phase HPLC?

Answer 9

A hydrophobic surface, often with C18 or C8 bonded groups.

Question 10

What is the primary role of the HPLC detector?

Answer 10

To sense and measure the presence of analytes in the mobile phase

Question 11

Name two types of detectors and their features.

Answer 11

UV-Vis Detector: Measures absorbance at specific wavelengths.
Refractive Index Detector: Universal detector but less sensitive.

Question 12

What is the main advantage of fluorescence detectors?

Answer 12

They are highly selective and sensitive (LOD ≈ 0.01 ng).

Question 13

List some pharmaceutical applications of HPLC.

Answer 13

Drug impurity testing.
Assays of drug substances/products.
Dissolution testing.
Bioanalytical testing (blood, plasma, urine).

Question 14

What does the area of a chromatographic peak represent?

Answer 14

The concentration of the analyte.

Question 15

What are the axes of a chromatogram?

Answer 15

X-axis: Retention time.
Y-axis: Absorbance (e.g., mAu).

Question 16

What are the advantages of HPLC?

Answer 16

High sensitivity and specificity.
Wide applicability for nonvolatile compounds.
Quantitative and qualitative analysis.

Question 17

What are the limitations of HPLC?

Answer 17

High cost of equipment and solvents.
Requires expertise to operate and interpret results

Question 18

What is the primary purpose of the stationary phase in chromatography?
A. To pump the mobile phase through the system.
B. To separate sample components based on their interactions with it.
C. To detect analytes in the sample.
D. To provide the sample injection point.

Answer 18

B

Question 19

What is the role of the guard column in HPLC?
A. To measure the concentration of analytes in the sample.
B. To remove components that may damage the analytical column.
C. To control the flow rate of the mobile phase.
D. To house the stationary phase used for separation.

Answer 19

B

Question 20

In reverse-phase HPLC, which of the following statements is TRUE?
A. The mobile phase is hydrophobic, and the stationary phase is hydrophilic.
B. Hydrophilic compounds are strongly retained by the stationary phase.
C. Hydrophobic compounds have stronger interactions with the stationary phase.
D. Water is a strong solvent in reverse-phase HPLC.

Answer 20

C

Question 21

Which detector type is known as the “universal detector”?
A. Fluorescence detector
B. UV-Vis detector
C. Refractive index detector
D. Electrochemical detector

Answer 21

C

Question 22

What does the area under a chromatographic peak represent?
A. The retention time of the analyte.
B. The concentration of the analyte.
C. The resolution of the analyte’s separation.
D. The polarity of the analyte.

Answer 22

B

Question 23

What does a retention time (
𝑡𝑅 ) in chromatography indicate?
A. The time it takes for the mobile phase to pass through the column.
B. The time a compound spends in the stationary phase.
C. The time from injection to detection of a compound.
D. The time needed for the guard column to filter impurities.

Answer 23

C

Question 24

What is the minimum resolution (
𝑅𝑠) value required for good separation in chromatography?
A. 0.5
B. 1.0
C. 1.5
D. 2.0

Answer 24

C

Question 25

Which of the following is a limitation of HPLC?
A. It is unable to analyze volatile compounds.
B. It provides low sensitivity for small samples.
C. It requires costly solvents and specialized equipment.
D. It cannot separate complex mixtures.

Answer 25

C

Question 26

What is the role of the HPLC pump?
A. To mix the sample with the mobile phase.
B. To deliver a precise and reproducible flow of the mobile phase.
C. To separate analytes in the stationary phase.
D. To measure the retention time of analytes.

Answer 26

B

Question 27

n a chromatogram, what does a Gaussian bell-shaped peak indicate?
A. Poor resolution of the analyte.
B. Analyte concentration is too high.
C. Ideal separation and symmetry of the analyte.
D. An error in the mobile phase composition.

Answer 27

C