HPLC
HPLC (High-Performance Liquid Chromatography) is a powerful analytical technique used for separating, identifying, and quantifying components in a mixture. It operates on the principles of liquid chromatography, with a liquid mobile phase carrying the sample through a stationary phase. The distinguishing feature of HPLC is the application of high pressure to enhance the separation efficiency, making it particularly suitable for complex pharmaceutical samples.
HPLC Applications in Pharmaceutical Industries:
1. Drug Development and Formulation: HPLC plays a pivotal role in drug development by providing detailed information about the purity, stability, and composition of pharmaceutical compounds. It is employed in the formulation stage to analyze raw materials, monitor reactions, and assess the final product’s quality.
2. Quality Control: Quality control is a critical aspect of pharmaceutical manufacturing, and HPLC is extensively used to verify the quality and consistency of drug formulations. It allows for the detection and quantification of impurities, ensuring that pharmaceutical products meet regulatory standards.
3. Assay Testing: HPLC is employed for assay testing to determine the concentration of the active pharmaceutical ingredient (API) in a drug formulation. Accurate quantification is essential to ensure that medications deliver the intended therapeutic effect.
4. Stability Testing: Pharmaceuticals must maintain their potency and stability over time. HPLC is utilized in stability testing to assess how the composition of a drug changes under various conditions, including temperature, humidity, and light exposure.
5. Identification of Unknown Compounds: In the complex world of pharmaceuticals, identifying unknown compounds is a common challenge. HPLC, often coupled with mass spectrometry (HPLC-MS), aids in the identification of unknown impurities or degradation products that may affect the safety and efficacy of medications.
HPLC Interview Questions
Question 1: What is the Full form of HPLC?
Answer: HPLC stands for High-performance Liquid Chromatography or High-pressure Liquid Chromatography.
Question 2: What is the HPLC Principle? What is the Principle of Chromatography?
Answer: It is a chromatographic technique used to separate the components from a mixture of a solution by using a solid stationary phase and a liquid mobile phase.
Question 3: What is the General chapter number of Chromatography?
Answer: USP General chapter Number <621>
Question 4: How many types of chromatography?
Answer: There are 12 types of chromatography.
Question 5: What are the names of different types of chromatography?
Answer:
3. Thin Layer Chromatography (TLC)
4. Paper Chromatography
5. Column Chromatography
6. Affinity Chromatography
7. Fast Protein Liquid Chromatography
8. Supercritical Fluid Chromatography
9. Reverse-phase Chromatography
10. Countercurrent Chromatography
11. Two Dimensional Chromatography
12. Pyrolysis Gas Chromatography
Question 6: What are the types of Column Chromatography?
Answer: There are 5 types of chromatography methods where the column is used.
1. Liquid Chromatography
2. Gas Chromatography
3. Ion Exchange Chromatography
4. Size Exclusion Chromatography
5. Chiral Chromatography
Question 7: What is the Column Chromatography Principle?
Answer: The column chromatography principle is that column chromatography is fundamentally based on the adsorption of solutes from a solution using a stationary phase, which then separates each component of the mixture.
Question 8: What is Reverse phase chromatography?
Answer: The mobile phase is more polar than the stationary phase is called reserve phase chromatography.
Question 10: What is Chiral Chromatography?
Answer: It is a chromatography to determine the content of chiral isomers whether it is in normal or reverse phase.
Question 11: What is a Column?
Answer: A column is a still tube that contains a stationary phase.
Question 12: What are the components of HPLC?
Answer: There are 6 types of components,
1. Reservoir
2. Pump
3. Sample Components
4. Column Components
5. Detector
6. Recorder
Question 13: What are the types of Detectors used in Chromatography?
Answer: There are 6 detectors used in HPLC based on their usage,
1. UV detector
2. PDA Detector
3. Fluorescence detector
4. Conductivity detector
5. Refractive Index Detector
6. Light Scattering Detector
Question 14: What is Gradient run?
Question 15: What is Isocratic run?
Answer: The mobile phase remains the same throughout the run. As mentioned above 20 min run time. The mixed (Composition of buffer + aqueous solution) mobile phase is used in the isocratic run.
Question 16: What is the Linear gradient?
Answer: The mobile phase remains unchanged throughout the run by using two different reservoirs.
Understand the below linear-gradient table. Mobile phase A and Mobile phase B is in the same proportion for up to 20 mins.
Question 17: What is Retention time (RT)?
Answer: Retention time is the time between injection and the appearance of the peak maxima.
Question 18: What is Relative retention time (RRT)?
Answer: RRT is a measure of the difference in affinities of two compounds for the stationary phase.
Question 19: Which calibration standard is used for HPLC Calibration?
Answer: Caffeine standard is used for HPLC calibration.
Question 20: Why caffeine standard is used for HPLC calibration?
3. Readily available in the market
4. It shows two maxima and one maxima at 205 nm,273 nm, and 245 nm
respectively.
Question 21: What type of material is most commonly used in the stationary phase?
Answer: Silica gel is the most commonly used material in the stationary phase.
Question 22: Why Silica gel is used in the stationary phase?
Answer: Silica gel is an inert material and does not react with the mobile phase.
Question 23: What is the Flow diagram of the HPLC System?
Answer:
1. Solvent reservoir
2. Pump
3. Degasser
4. Mixing valve
5. Guard column
6. Sample injector
7. Column
8. Detector
9. Recorder
10. Outlet
