Chromatography 2 Flashcards
What is chromatography?
A technique to separate, identify, and quantify components in a mixture by passing them through a stationary phase.
What are the two main outputs of chromatography?
Qualitative analysis: Identifies the components (e.g., retention time).
Quantitative analysis: Measures the concentration (e.g., peak area).
What are the six key components of an HPLC system?
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.
What is the role of the guard column in HPLC?
It removes sample components that may foul the analytical column, prolonging its lifespan.
What is retention time (
π‘π
) in HPLC?
The time it takes for an analyte to travel from the injector to the detector.
What is dead time (
π‘0 ) in HPLC?
The time required for an inert compound to pass through the column without interacting with the stationary phase.
How is resolution (
π
π ) calculated in HPLC?
Good separation requires
π
π β₯1.5.
What are the two types of column packing?
Pellicular particles: Porous stationary phase on a solid core; used in guard columns.
Porous particles: Silica or polymer particles; used in analytical columns.
What is the stationary phase in reverse-phase HPLC?
A hydrophobic surface, often with C18 or C8 bonded groups.
What is the primary role of the HPLC detector?
To sense and measure the presence of analytes in the mobile phase
Name two types of detectors and their features.
UV-Vis Detector: Measures absorbance at specific wavelengths.
Refractive Index Detector: Universal detector but less sensitive.
What is the main advantage of fluorescence detectors?
They are highly selective and sensitive (LOD β 0.01 ng).
List some pharmaceutical applications of HPLC.
Drug impurity testing.
Assays of drug substances/products.
Dissolution testing.
Bioanalytical testing (blood, plasma, urine).
What does the area of a chromatographic peak represent?
The concentration of the analyte.
What are the axes of a chromatogram?
X-axis: Retention time.
Y-axis: Absorbance (e.g., mAu).
What are the advantages of HPLC?
High sensitivity and specificity.
Wide applicability for nonvolatile compounds.
Quantitative and qualitative analysis.
What are the limitations of HPLC?
High cost of equipment and solvents.
Requires expertise to operate and interpret results
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.
B
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.
B
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.
C
Which detector type is known as the βuniversal detectorβ?
A. Fluorescence detector
B. UV-Vis detector
C. Refractive index detector
D. Electrochemical detector
C
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 analyteβs separation.
D. The polarity of the analyte.
B
What does a retention time (
π‘π
) 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.
C
What is the minimum resolution (
π
π ) value required for good separation in chromatography?
A. 0.5
B. 1.0
C. 1.5
D. 2.0
C
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.
C
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.
B
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.
C
