Troubleshooting HPLC: Basic to Advance Level Interview Questions with Answers

Here are some common HPLC interview question from basic to advace level with troubleshooting. For detail information about HPLC try to visit this post:- HPLC Made Easy: Understanding Principle, Instrumentation, and System Suitability

1. Whatis HPLC?

HPLC stands for High Performance Liquid Chromatography. It is an analytical technique usd to separate, identify,quantify components in a mixture. It is mostly used in pharmaceutical, cosmetic, food industry, enviromental testing.

2. What is the principle of  HPLC?

HPLC principle based on adsorption or partition chromatography. It  acts according to interaction of sample with bot stationary phase and mobile phase. Compounds having stronger affinity with stationary phase will moves slower, on other hands compounds which have stronger affinity with mobile phase thats moves 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 mobile phase phase.
• Degasser: To remove dissolved air in mobile phase
• Pump: It delivers M.P. toward the column at constant flow rate.
• Injector: Helps to inject specified amount of  sample in the mp which passes through the column.
• Column Oven: It helps to store the column at mentioned temperature.
• Column:It is stationary phase that helps to separate component presents in  the mixture.
• Detectors: Detects the compounds separated in the column.
• Data System: It's analyse and records the chromatographic data.

4. How normal phase is different from reverse phase?

Components	Normal phase	Reverse phase
Stationary Phase	More Polar (e.g. silica)	Non-polar (e.g. C18, C8)
Mobile Phase	Non Polar (e.g. Hexane)	polar (e.g. Water, Methanol)
Separates	Non-polar compounds	Polar & Non-polar (Mostly used)

5. Describe 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 compounds.

6. What do you mean by Mobile Phase?

Mobile phase is the liquid solvent that carries the sample and move towards the column. It may be a single solvent or mixture of two or more solvents according to their chemical properties.

7. What is Isocratic Elution in HPLC?

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

8. What is gradient elution?

In gradient elution, the composition of mobile phase ratio gradually change at different time during complete analysis. Here compounds having wide properties can be separated.

9.  What are the varous types og 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 pump	Mixing occurs in mixing chamber, solvents of both pump meet
Require normal pressure or low-pressure	Require High-pressure.

10. Which types HPLC Column available in the maarket?

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

11. What are the different types of detector 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 Scatting Detector (MALSD)
• Radioactivity detector
• NMR detector
• Electrochemical detector
• HPLC conductivity deector

12.  What are the most commonly used detector in HLPC?

UV detector and PDA-Photodiode array detector

13. What is the use of Guard column?

It protects the main  analytical column from contaminants, particulate matter, and any impurities which can decrease it's perforance.. It increases the durability of Column.

14. How UV Visible detector are differ from PDA detector?

A UV Visible detector measure absorbance at sigle wavlength. making it ideal for analyte with known absorbance maxima.

PDA detectors measure absorbance across a range of wavelength simultaneously.

15.  What is the general Chalpter for HPLC in USP?

The general chapter 621 is for HPLC in USP.

16. What are the diffrence between C18 and C8 Column?

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	Separate more non-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

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

Column Efficiency describe the performance of the column. It is expressed as the number of theoretical plate. If there is higher column efficiency then column separate component properly and sharp, better peaks.

18. What are the HPLC start up procedure?

i. First of all check any leaks, damage or loose fitting of tube, electricity connection.

ii. Waste Bottle should be empty.

iii. Prepare fresh mobile phase as per MOA.

iv. Filter the mobile phase though vaccum filter using 0.45 um filter and sonicate about five minutes.

v. Label the all mobile phase properly.

vi. Select the correct column and connect properly.

vii. Power on the HPLC components like Degasser, pump, autosampler, column oven, detector, software.

viii. Connect intrument with software.

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

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

xi. Load the method and allow the system to equilibrate about 30 minutes to get stable base line.

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

xiii. Run blank sample to ensure contamination.

xiv. Single run a standard to conform 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 are the HPLC shutdown procedure?

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

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

iii. Store the column.

iv. Turn off the components reverse-wise like detecter, column oven, auto sampler, pump, degasser.

20.What is system sutability?

System sutability is the checking of system before, during the analysis to conform system performance,

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

21. How to use new reverse phase column first time?

When you install new reverse phase column then flush the column with acetonitrile or methanol about 1 hours. Increase the flow rate lower to higher slowly.

22. What is chromatogram?

Chromatogram is the grahical representation of detector response or quantities measured value vs time. It conatins peaks on the baseline.

23. What is the retention time in chromatography?

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

24. What is Dwell Volume?

The dwell volume is the volume between the point where mobile phase meet and 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 like air or 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 X Flow rate (ml per minute).

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

Buffer helps to control pH, also improve reproducibility and helps to get better peak shape by reducing component interaction with stainary phase.

28. How can you improve peak resolution in HPLC?

• Maintain the buffer pH properly and mobile phase composition correctly.
• Coumn having smaller particle size should ne used.
• Adjust flow rate and temperature propely.
• Use the column having longer length.

29. How do you baseline drift in HPLC?

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

30. What are the cause 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 sample is overloaded.

31. What is 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 be not 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 resoluion in chromatography?

The formula for electronic calculation

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

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

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 as theoretical plate which describe the column efficiency. Higher the theoretical plate number more performance of column is achieved.

What are 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 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 remain in HPLC system after analysis that can contaminate other samples. To minimize it clean the injector properly, optimize sample prepration, washing properly.