HPLC (Questions & answers)
Q.1- What is the difference between C8 and C18?
Answer: – C8 has 8 carbon atoms and C18 has 18 carbon atoms, C18 is more hydrophobic than C8 due to that C18 has a long retention time and C8 has a sort retention time (early).
Q.2- What is the difference between related substances and Chromatographic purity?
Answer: Related substance is used to determine known as well as unknown impurities while chromatography purity is used to determine only known impurities from the sample.
Q.3- What is the baseline in HPLC?
Answer: Baseline is a detector response to the mobile phase.
Q.4- Why buffer is used in the mobile phase?
Answer: Buffer is used resisted change in PH.
Q.5- Why guard column used in HPLC?
Answer: To protect the analytical column from contamination.
Q.6- What is the difference between a stationary phase and the mobile phase?
Answer: Stationary phase does not move with the sample it sticks in the column. While the mobile phase moves with the sample throughout the column.
Q.7- What is the carry-over in HPLC?
Answer: Void volume is a volume of mobile phase required to elute an un-retained peak.
Q.8- What is the dead volume in HPLC?
Answer: Dead volume is the volume of the HPLC system between points of injection to point of detection.
Q.9- What is dwell volume in HPLC?
Answer: Dwell volume is a volume of gradient HPLC system between mixing chamber in column inlet.
Q: 10- What are the most buffer used in HPLC?
Answer: Acetate buffer, Phosphate buffer are the most common buffers used in HPLC.
Q.11- How should I store the HPLC column?
Answer: Normal phase column can be stored in ethanol and the reverse phase column is stored in a mixer of water and organic solvent (ANC/ Methanol).
Q.12- What is the maximum pressure in HPLC?
Answer: It depend on which make you are using for water HPLC 5000 psi.
Q.13- What is the USP general chapter number for chromatography?
Answer: USP general chapter number <621>.
Q.14- How many types of chromatography?
Answer: Two types of chromatography
- Liquid chromatography
- Gas chromatography.
Q.15- What is the full form of HPLC?
Answer: High-pressure liquid chromatography is high-performance liquid chromatography.
Q.16- Why are we called high-pressure liquid chromatography?
Answer: Because applying high pressure by using pumps to chromatography.
Q.17- What is the principle of HPLC?
Answer: HPLC is a separation technique of components from the mixer by using a solid stationary phase and a liquid mobile phase.
Q.18- What is reverse phase chromatography?
Answer: Where the mobile phase is more polar than the stationary phase is called reverse chromatography.
Q.19- What is the normal phase chromatography?
Answer: Where stationary phase is more polar than the mobile phase as called normal chromatography.
Q.20- What is the column?
Answer: A still tube that contains a stationary phase.
Q.21- What are the component of HPLC?
Answer:
- Reservoir
- Pump
- Sample component
- Column components
- Detector
- Recorder.
Q.22- What are types of detectors used in HPLC?
Answer:
- UV detector
- PDA
- Fluorescent detector
- Conductivity detector
- Reflective detector
- Light scattering detector.
Q.23- What is the gradient run.
Answer: The mobile phase composition varies over run time
| RT | A | B |
| 1 | 30 | 70 |
| 2 | 40 | 60 |
| 3 | 50 | 50 |
| 4 | 60 | 40 |
Q.24- What is an isocratic run?
Answer: Mobile phase (mixed MP) remains the same throughout the run.
Exp. from 1min to 4min, it’s the same.
Q.25- What is the linear gradient?
Answer: Mobile phase remains the same throughout the run by using two different reservoirs.
Ex.
| RT | A | B |
| 1 | 30 | 70 |
| 2 | 30 | 70 |
| 3 | 30 | 70 |
| 4 | 30 | 70 |
Q.26- What is the retention time?
Answer: It is a time between injection and the appearance peak maxima.
Q.27- What is the relative retention time?
Answer: It is a measure of the difference of affinities of two compounds for the stationary phase.
Q.28- Which standard we used for HPLC calibration?
Answer: Caffeine standard used for HPLC calibration.
Q.29- Why is caffeine standard used for calibration?
Answer:
- It is very stable
- It is durable
- Readily available in the market.
- It shows two maxima one minimum at 205, 273, and 245 nm respectively.
Q.30- What is the most commonly used stationary phase?
Answer: Silica gel is used in the stationary phase.
Q.31- Why is silica gel used in the stationary phase?
Answer: Silica gel is an inert material and doesn’t react with the mobile phase.
Q.32- Explain the flow diagram of the HPLC system?
Answer:
- Solvent reservoir to store MP
- Pump to move MP in pressure.
- Degasser to remove dissolves air.
- Mixing volume to mix MP
- Guard column to protect column.
- Sample injector to inert sample.
- Column to separates components.
- Detector to detect the component.
- Recorder to record the analyzed data.
- Outlet to collect waste.
