HPLC Interview Questions with Answers

Following are the top HPLC Interview Questions with Answers:

What is the full-form of the HPLC?

The full-form of HPLC is high-performance liquid chromatography or high-pressure liquid chromatography

What is the principle of HPLC?

HPLC (high-pressure/high-performance liquid chromatography) is a separation technique based on solid stationary and liquid mobile phases. Separations depend upon the polarity of the stationary phase, mobile phase and molecules. Separation may be achieved by either the Partition mechanism or the Adsorption mechanism

What are the different components of HPLC?

The following are the different components of HPLC:

• Mobile Phase
• Mobile phase reservoir
• Degassers
• Pump
• Mixing valve
• Guard column
• Sample injection port
• Injector
• Column
• Column temperature controller
• Detector
• Waste Collector
• Data processor &
• Chromatogram

What is the guard column in HPLC and what is its role?

The guard column is a short column that contains a same stationary phase as of the analytical column. But in the guard is filled with bigger particles size to avoid any unnecessary pressure. It removes the impurities present in the buffer and solvents of the mobile phase.

What is the HPLC column?

The HPLC column is the heart of the HPLC and it is responsible for the separation of different components. It is packed with the stationary phase like C18, C8 phase.

Which column is the universal column in HPLC?

Cyano column is universal column in HPLC

What is the mobile phase?

Mobile phase in HPLC is solvent or mixture of solvents or mixture of solvents containing solid buffers.

What is Isocratic mode of elution?

In the isocratic mode of elution composition of solvent in the mobile phase does not change or remain constant. For example if the mobile phase is mixture of water and acetonitrile in a composition of 60:40. It means this composition will remain the same throughout the analysis.

What is the Gradient mode of elution?

In the gradient mode of composition of solvent changes with time. It is used to elute non-polar compound. For example:

What is the difference between NPC (Normal phase chromatography) and RPC (Reverse phase chromatography)?

In RPC, the mobile phase is polar e.g., a mixture of Water/Buffer and organic solvents like acetonitrile, methanol, ethanol, IPA, THF etc and the stationary phase is non-polar or less polar e.g., C18 (ODS), C8, Cyno etc. The sample should be soluble in water or in a mixture of water and organic solvents.

What will be the elution order of Toluene and Benzoic acid in RPC mode?

First Benzoic acid will elute and then after Toluene will elute.

What are the different applications of HPLC?

The HPLC is used in both qualitative (identification test) and quantitative analysis in the following industries:

• Pharmaceuticals
• Food
• Testing Lab
• Research centres
• Biotech Industries
• Pesticide Industries

What are the different types of detectors used in HPLC?

The following detectors are used in HPLC analysis:

• Ultraviolet/Visible Absorbance (UV/Vis)
• Mass Spectrometer (MS)
• Refractive Index (RI)
• Evaporative Light Scattering (ELS)
• Fluorescence (FL)
• Electrochemical (EC)

What are the criteria for selecting a detector?

The selection of a Detector  is based on:

• Chemical nature of analytes and potential interferences
• Limit of detection
• Availability and/or cost of the detector

What are the advantages and disadvantages of RPC?

The following are the advantages and disadvantages of Reverse phase chromatography:

Advantages:

• Most commonly used chromatography
• Longer column life
• Lesser system equilibration time
• LC-MS compatible method can be developed
• Work well for the weak, acid, weak base and non-polar molecules
• Order of elution is hydrophobic to hydrophilic

Disadvantages:

• Does not work for strongly ionized compounds e.g. Strong acidic compounds and strong Basic compounds

Why is the mobile phase filtered out?

The following are the main reasons for filtering the mobile phase:

• During the preparation of most of the mobile phase, solid chemicals like K₂HPO₄, KH₂PO₄, Na₂HPO₄, and NaH₂PO₄ etc. are used. These solid chemicals are dissolved in water during buffer preparation. These chemicals may contain water-insoluble particles as impurities and can cause problems during HPLC analysis such as noise, and column choking.
• Secondly, during the preparation of the mobile phase, two or more solvents are mixed, and due to this mixing air also dissolves in the mobile phase. N₂ (Nitrogen) and O₂ (Oxygent) in the air have UV absorption so they give their respective peaks as noise.
• Dissolved air can also cause a drop in the pressure during analysis.
• That is why the mobile phase is filtered out during HPLC analysis

What is the general chapter of chromatography in USP?

The general chapter of chromatography in <USP is 621>

Which standard is used for the calibration of HPLC?

Caffeine or Uracil can be used for calibration of HPLC

Why Caffeine is used for the calibration of HPLC?

Caffeine is the stable molecule and readily available in pure form and that is why caffeine is used for the calibration of HPLC

How to decrease retention time in HPLC?

• Increasing organic solvents such as methanol, and acetonitrile or decreasing aqueous solvents such as water or buffer reduces retention time in reverse phase HPLC.
• Decreasing nonpolar organic solvents or decreasing polar organic solvents reduces retention time in normal phase HPLC.

What increases retention time in HPLC?

• Decreasing organic solvents such as methanol, and acetonitrile or increasing aqueous solvents such as water or buffer increases retention time in reverse phase HPLC.
• Increasing nonpolar organic solvents or decreasing polar organic solvents increases retention time in normal phase HPLC.

What causes HPLC retention time shifts?

Retention time may shift due to change due to the following reasons:

• Change in the composition of the mobile phase
• Change in the column temperature
• Change in flow rate
• Improper column equilibration
• If bubbles trap in the tubing

Does temperature affect retention time in HPLC?

Yes. On increasing temperature retention time is decreased and on decreasing temperature retention time is increased

Why does the HPLC retention time change for each run?

Due to pressure fluctuation or improper column equilibration retention time may change in each run

Conclusion

I hope this article has helped you to clear your doubts about HPLC. Now you can independently handle the HPLC more effective way. You may also want to check out other articles on my blog such as how to understand the principle of HPLC.

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