Troubleshooting HPLC: Basic to Advance Level Interview Questions with Answers

Here are some common HPLC interview questions, from basic to advanced, with troubleshooting. For detailed information about HPLC, try visiting this post: HPLC Made Easy: Understanding Principle, Instrumentation, and System Suitability.

1. What is HPLC?

HPLC stands for High-Performance Liquid Chromatography. It is an analytical technique used to separate, identify, and quantify components in a mixture. It is mostly used in the pharmaceutical, cosmetic, and food industries, as well as in environmental testing. 

2. What is the principle of HPLC?

HPLC is a principle-based method based on adsorption or partition chromatography. It acts according to the interaction of the sample with both the stationary phase and the mobile phase. Compounds with stronger affinity for the stationary phase move more slowly; on the other hand, compounds with stronger affinity for the mobile phase move faster through the column and get separated first. 

3. What are the main components of HPLC?

The main components of HPLC are

• Solvent reservoir: To store the mobile phase.
• Degasser: To remove dissolved air in the mobile phase
• Pump: It delivers the mobile phase toward the column at a constant flow rate.
• Injector: Helps to inject a specified amount of sample into the MP, which passes through the column.
• Column Oven: It helps to store the column at the mentioned temperature.
• Column: It is a stationary phase that helps to separate components present in the mixture.
• Detectors: Detect the compounds separated in the column.
• Data System: It analyses and records the chromatographic data.

4. How is the normal phase different from the reverse phase?

Components	Normal phase	Reverse phase
Stationary Phase	More Polar (e.g., Nonpolar)	Nonpolar (e.g., C18, C8)
MobilNonpolar	Nonpolar (e.g., Hexane)	Polar (e.g., water, methanol)
SeNonpolar	Nonpolar compounds	Nonpolar-polar (mostly used)

5. Describe the stationary phase.

The stationary phase is a column that is packed with materials like silica or polymer particles. It helps to separate the components after interaction. Separation depends upon the affinity of the compound. 

6. What do you mean by mobile phase?

The mobile phase is the liquid solvent that carries the sample and moves towards the column. It may be a single solvent or a mixture of two or more solvents, depending on their chemical properties. 

7. What is isocratic elution in HPLC?

In an isocratic system, the composition of the mobile phase is constant throughout the whole analysis. When the compounds have the same properties, this method is suitable for analysis. 

8. What is gradient elution?

In gradient elution, the composition of the mobile phase ratio gradually changes at different times during the complete analysis. Here, compounds with wide properties can be separated. What are the various types of gradient mixing?

Low-pressure gradient	High-pressure gradient
Mixing of mobile phase before pump	Mixing of mobile phase after pump
Mixing occurs at the side of the device before the pump.	Mixing occurs in the mixing chamber; solvents of both pumps meet.
Requires normal pressure or low pressure	Requires high pressure.

0. Which types of HPLC columns are available in the market?

• C18: Octadecylsilane Column
• C8: Octylsilane Column
• Ion Exchange Column
• Size-exclusion Column
• Chiral Column

1. What are the different types of detectors for HPLC?

• UV-Visible detector (UVVis)
• PDA detector (Photodiode Array)
• Fluorescence detector
• Refractive Index detector (RI)
• Mass spectrometry detector
• Evaporative Light Scattering Detector (ELSD)
• Multi-angle Light Scattering Detector (MALSD)
• Radioactivity detector
• NMR detector
• Electrochemical detector
• HPLC conductivity detector

. What are the most commonly used detectors in HPLC?

UV detector and PDA (photodiode array detector)

3. What is the use of a guard column?

It protects the main analytical column from contaminants, particulate matter, and any impurities that can decrease its performance. It increases the durability of the column.

4. How are UV-visible detectors different from PDA detectors?

A UV-visible detector measures absorbance at a single wavelength. This makes it ideal for analytes with known absorbance maxima.

PDA detectors measure absorbance across a range of wavelengths simultaneously.

5. What is the general chapter for HPLC in USP?

General Chapter 621 is for HPLC in the USP.

6. What are the differences between C18 and C8 columns?

Feature	C18 (Octadecylsilane)	C8 (Octylsilane)
Bonded phase	18 carbon atoms (longer chain)	8 carbon atoms (shorter chain)
Hydrophobicity	More hydrophobic	Less Hydrophobic
Retention time	Longer	Shorter
Polarity of analytes	Separation of nonpolar-polar compounds	Good for moderately polar compounds
Elution time	Slower	Faster
Resolution	Higher for complex mixture	Lower for highly hydrophobic compounds
Usage	More common & versatile	Used for faster analysis

7. What do you mean by column efficiency in HPLC?

Column efficiency describes the performance of the column. It is expressed as the number of theoretical plates. If column efficiency is higher, the column separates components properly and gives sharper peaks.

8. What is the HPLC start-up procedure? 

i. First of all, check for any leaks, damage, or loose fittings of the tube and electrical connection.

ii. The waste bottle should be empty. 

i i. Please prepare fresh mobile phase as per MO 

v. Filter the mobile phase through a vacuum filter using a 0.45 um filter and sonicate for about five minutes. 

v. Label all mobile phases properly.

i. Select the correct column and connect properly.

v i. Power on the degasser components like the degasser, pump, autosampler, column oven, detector, and software.

viii. Connect the instrument to the software.

ix. Open the purge valve and start the pump valve at a low flow rate through the software.

x. Purge each solvent for about 5-10 minutes to remove air bubbles. Close the purge valve.

xi. Load the method and allow the system to equilibrate for about 30 minutes to get a stable baseline.

xii. Create a batch file or path where you want to save the data.

xiii. Run a blank sample to ensure contamination.

xiv. Single-run a standard to confirm system suitability, like theoretical plate, tailing factor, resolution, retention time, peak shape.

xv. Load the sample and set sequences serially: blank, standard, test, and run.

19. What is the HPLC shutdown procedure?

i. Wash the column properly after analysis completion for about 30 minutes. 

ii. For washing, a 50:50 ratio of acetonitrile and water is used.

iii. Store the Column.

iv. Turn off the components in reverse order: detector, column degasser, servo sampler, pump, degasser

20. What is system suitability?

System suitability is checking the system before and during the analysis to confirm system performance.

Different parameters like retention time, column efficiency, resolution, and peak tailing are checked.

21. How to use a new reverse-phase column for the first time?

When you install a new reverse-phase column, flush the column with acetonitrile or methanol for about 1 hour. Increase the flow rate from lower to higher slowly.

22. What is a chromatogram?

A chromatogram is the graphical representation of detector response or measured quantities vs. time. It contains peaks on the baseline.

23. What is the retention time in chromatography?

Retention time is the time between the injection of the sample and the maximum peak response after elution.

24. What is Dwell Volume?

The dwell volume is the volume between the point where the mobile phase meets the inlet or top of the column.

25. What is hold-up time in liquid chromatography?

The required time for the elution of an unretained compound, such as air or an unretained solvent peak, with the baseline scale in minutes.

26. What do you mean by hold-up volume in chromatography?

The required volume of mobile phase for elution of an unretained compound.

It is calculated as hold-up time × flow rate (ml per minute).

27. What is the role of a buffer in the mobile phase?

Buffer helps control pH, improve reproducibility, and improve peak shape by reducing component interaction with the stationary phase.

28. How can you improve peak resolution in HPLC?

• Maintain the buffer pH properly and mobile phase composition correctly.
• Use a column with a smaller particle size.
• Adjust flow rate and temperature properly.
• Use a column with a longer length.

29. How do you baseline drift in HPLC?

• First, ensure the mobile phase is not contaminated.
• Try to prepare a new mobile phase.
• Degas or sonicate the solvent.
• Change or clean the column properly.
• Maintain the column temperature.

30. What are the causes of peak tailing in HPLC?

• Due to poor column condition or overuse.
• Strong affinity between analyte and stationary phase.
• Mobile phase pH is not maintained properly.
• When the sample is overloaded. 

31. What is the tailing factor or symmetry factor in HPLC?

It is the distance from the front slope of the peak to the back slope divided by twice the distance from the center line of the peak to the front slope. The general limit is from 0.8 to 1.8. It should not be more than 2.

32 What do you mean by resolution in HPLC?

Resolution is the separation of two components in the mixture. It is the distance between the two peaks.

What is the formula to calculate resolution in chromatography?

The formula for electronic calculation

R=1.18 (TR2-TR1)/(Wh1-Wh2)

WhereTR1 = retention time of the peak eluting first
TR2 = retention time of the second peak eluting; Wh1 = peak width at half height of the first peak
Wh2 = Peak width at half height of the second peak

The formula for manual calculation

R=2.0 (TR2-TR1)/(Wb1-Wb2)

Where TR1 = Retention time of the peak eluting first
TR2 = Retention time of the second peak eluting

Wb1 = Peak width at the base of the first peak

Wb2 = Peak width at the base of the second peak

33. What is plate number in chromatography?

It is also called the theoretical plate, which describes the column efficiency. The higher the theoretical plate number, the better the performance of the column

What is the formula to calculate the theoretical plate?

The formula for electronic calculation is 

N = 5.54 (Tr/Wh)²

Where Tr = retention time of the peak corresponding to the component.

Wh = Peak width at half height of the peak

For manual calculation, the formula given below is used

N = 16 (Tr/Wb)²

Where Tr = Retention time of the peak corresponding to the component.
Wb = Peak width at the base of the peak

34. What is carryover in HPLC?

Carryover is a residual sample remaining in the HPLC system after analysis that can contaminate other samples. To minimize it, clean the injector properly, optimize sample preparation, and wash properly.