Chromatography 1 Flashcards
What is chromatography?
A technique to separate components of a mixture by passing them through a stationary phase, based on properties like size, charge, hydrophobicity, and affinity.
What are the two primary goals of chromatography
Preparative chromatography: Isolate sufficient quantities of a target component.
Analytical chromatography: Quantify and analyze individual components.
What does HPLC stand for?
High-Performance Liquid Chromatography.
What are the two main modes of mobile phase composition in HPLC?
Isocratic: Mobile phase composition remains constant throughout.
Gradient: Mobile phase strength increases over time to improve separation.
What is the role of the HPLC pump?
To deliver a precise, reproducible, constant, and pulse-free flow of the mobile phase.
What are the main characteristics used to separate compounds in HPLC?
Polarity (normal-phase and reverse-phase chromatography)
Electrical charge (ion-exchange chromatography)
Molecular size (size exclusion chromatography)
Affinity (affinity chromatography)
How does reverse-phase HPLC (RP-HPLC) work?
Stationary phase: Hydrophobic.
Mobile phase: Polar.
Nonpolar compounds interact more with the stationary phase and elute slower.
What is size exclusion chromatography?
A technique that separates molecules based on size. Smaller molecules penetrate the pores of the stationary phase and elute slower than larger molecules.
How does solvent strength affect separation in HPLC?
Stronger solvents elute solutes faster, reducing retention time.
In RP-HPLC, water is a “weak” solvent, and organic solvents are “strong.”
How does pH affect retention in RP-HPLC?
Low pH suppresses ionization of weak acids, increasing retention.
Ionized compounds interact less with the hydrophobic stationary phase.
What is the typical flow rate in HPLC?
0.5–2 mL/min.
What is the difference between a chromatograph and a chromatogram?
Chromatograph: The instrument used in chromatography.
Chromatogram: The graphical output showing separated components.
What is the role of the HPLC injector?
To introduce a precise sample volume into the mobile phase.
List pharmaceutical applications of HPLC.
Identification and assay of drug substances.
Impurity and preservative testing.
Dissolution testing.
Bioanalytical testing (blood, plasma, urine).
What is the purpose of HPLC in drug impurity testing?
To identify and quantify trace impurities in pharmaceutical formulations.
What are common challenges in HPLC analysis?
Gas bubbles causing band broadening.
Interference from impurities.
High cost of instrumentation and solvents.
What are common methods of solvent degassing?
Vacuum filtration.
Helium purging.
Ultrasonication.
What is the main principle of chromatography?
A. Mixing components of a sample to ensure uniformity.
B. Separating components based on their interaction with a stationary phase.
C. Heating components to their boiling points for analysis.
D. Using electrical fields to sort molecules by charge.
B
In reverse-phase HPLC (RP-HPLC), which type of solvent is considered “strong”?
A. Water
B. Organic solvents
C. Buffers
D. Nonpolar solvents like hexane
B
What is the purpose of a gradient mobile phase in HPLC?
A. To maintain a constant solvent composition during separation.
B. To improve separation by increasing solvent strength over time.
C. To reduce solvent consumption during analysis.
D. To enhance sample stability during elution.
B
Which separation mechanism is used in size exclusion chromatography?
A. Molecular polarity
B. Electrical charge
C. Molecular size
D. Affinity for specific ligands
C
What does the chromatogram represent in HPLC?
A. The instrument used for chromatography.
B. A graphical output showing separated sample components.
C. The stationary phase used in the column.
D. A type of mobile phase used in the process.
B
Which type of ion-exchange chromatography would you use to separate positively charged molecules?
A. Strong cation exchanger
B. Strong anion exchanger
C. Size exclusion column
D. Affinity chromatography
a
What effect does increasing the flow rate in HPLC have?
A. Increases retention time and resolution.
B. Reduces retention time and analysis time.
C. Improves the sensitivity of the detector.
D. Reduces the efficiency of solvent degassing.
B
What is the role of solvent degassing in HPLC?
A. To remove solid particles that can foul the column.
B. To prevent gas bubbles that interfere with detection.
C. To improve the solubility of the analyte in the mobile phase.
D. To ensure a constant flow rate in the pump.
B
Which of the following is a limitation of HPLC?
A. High sensitivity for trace analysis.
B. Wide applicability to non-volatile compounds.
C. Requires costly solvents and instrumentation.
D. Ability to separate complex mixtures efficiently.
C
How does pH influence retention in RP-HPLC?
A. High pH increases retention of weakly acidic drugs.
B. Low pH decreases retention of weakly basic drugs.
C. Low pH suppresses ionization of weak acids, increasing retention.
D. High pH suppresses ionization of weak acids, decreasing retention.
C
