81. What are the solutions to prevent the issue of Low Back Pressure in HPLC systems?
Leakage in the HPLC system – Identify the leakage and correct it
Column temperature is higher than required – Set the temperature as per method requirement
Flow rate is lower than required – Set the flow rate as per the method requirement
82. What are the good habits that help to minimize the HPLC system related issues such as peak shape problems, retention time variation, ghost peaks and column back pressure issues?
For new projects, select well researched, high-purity silica-based column and use highest quality HPLC-grade reagents.
Flush the HPLC system at regular intervals that removes salts and buffers.
Service the system periodically to reduce check-valve and pump-seal problems.
Precise sample preparation with adequate filtration and sample clean-up process reduces sample related issues.
Strong-solvent flush after every run or at specific frequency will reduce sample carryover and extend column lifetimes.
Columns won’t last forever, but with proper care, you should be able to get a good return on your investment.
83.What precautions should be considered before the first sample run in the HPLC?
i. Once the mobile phase is prepared and run in the system, leave the HPLC system for a little time to equilibrate to flush impurities or dirt if any out of the column.
ii. Dissolve samples adequately as per method of analysis.
iii. No particles should remain in the sample, use membrane filtration as per method.
iv. Ensure that your dissolved sample should not precipitate in the mobile phase.
v. Inject a standard and take a look at the chromatogram.
vi. Ensure that the baseline is stable with no drift and peaks are symmetrical.
vii. Ensure that the chromatogram after the second injection is identical to the first one.
84. What are the various types of detectors used in HPLC systems?
(i) UV-Visible HPLC Detector
(ii) PDA Detectors (Diode Array Detector or Photo Array Diode Detector) HPLC detector
(iii) Refractive-Index HPLC Detector
(iv) Evaporative Light Scattering Detector (ELSD) HPLC Detector:
(v) Multi-Angle Light Scattering Detector (MALS) for HPLC:
(vi) HPLC-Mass Spectrometer (HPLC-MS) Detector
(vii) HPLC Conductivity Detector:
(viii) HPLC-Fluorescence Detector:
(ix) Chemiluminescence HPLC Detector:
(x) HPLC Optical Rotation Detector or Chiral Detector
(xi) Electrochemical (Amperometric) Detector for HPLC
(xii) HPLC Photoconductivity Detectors
(xiii) HPLC Infrared (IR) Detectors
(xiv) Laser-Induced Fluorescence Detector
(xv) Radioactivity Detector
(xvi) HPLC-NMR Detector
85. 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.
86. 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.
87. 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.
88. What is the mobile phase?
Mobile phase in HPLC is solvent or mixture of solvents or mixture of solvents containing solid buffers.
89. 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.
90. 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.
91.What is 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.
92. 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
93. 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)
94 . 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
95. 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
96. 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.
97. What is the general chapter of chromatography in USP?
The general chapter of chromatography in USP is <621>
98. 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.
99. 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.
100. 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
101. Does temperature affect retention time in HPLC?
Yes. On increasing temperature retention time is decreased and on decreasing temperature
retention time is increased
Q. Which column is used in RP-HPLC (Reverse Phase High-Performance Liquid Chromatography)?
In Reverse Phase HPLC, the stationary phase is non-polar and the mobile phase is polar.
The most commonly used columns are C18 columns, known for their strong hydrophobic nature.
1. C18 Column (Octadecylsilane – ODS)
Most widely used in RP-HPLC.
Contains 18 carbon alkyl chains bonded to silica particles.
Provides high retention for non-polar to moderately polar compounds.
Suitable for pharmaceuticals, APIs, impurities, and natural products.
2. Other Common Columns
C8 (Octylsilane):
Shorter carbon chain than C18 → less retention.
Used for faster elution or moderately non-polar compounds.
Phenyl Column:
Contains aromatic rings → used for aromatic or π–π interaction compounds.
Cyano (CN) Column:
Offers intermediate polarity and different selectivity for polar analytes.
3. Column Specification
Typical dimension: 250 mm × 4.6 mm, particle size 3–5 µm.
Made of stainless steel or PEEK with silica-based stationary phase.
Q. Why do we use a C18 column instead of a C8 column in RP-HPLC?
C18 is preferred because it offers stronger retention, better resolution, and wider applicability — making it the universal choice for RP-HPLC analysis.
1. Longer Carbon Chain (More Hydrophobicity):
C18 has 18 carbon atoms compared to 8 in C8, making it more non-polar.
Provides stronger interactions with non-polar analytes → better separation and retention.
2. Higher Retention Time:
C18 retains compounds longer, allowing better resolution of closely eluting peaks.
Ideal for complex mixtures where complete separation is required.
3. Broader Application Range:
C18 can separate non-polar, moderately polar, and some polar compounds effectively.
Used widely in pharmaceutical, food, and chemical analysis due to versatility.
4. Better Reproducibility and Peak Shape:
Provides consistent retention and sharp peaks, improving method reliability.
5. Stability and Popularity:
Most validated and standardized column in pharmacopoeial methods (USP/EP).
Easier to compare and reproduce results across labs.
When to use C8:
Used when shorter retention or faster analysis is needed, or when compounds are very hydrophobic.
Q: What is the acceptance criterion for flow rate accuracy in HPLC?
Flow rate accuracy ensures the pump delivers the correct solvent volume per unit time.
As per HPLC calibration standards, the flow rate should be within ±2.0% of the set value.
It verifies the precision of solvent delivery, ensuring reproducible retention times and consistent chromatographic performance.
Accurate flow rate is critical for maintaining method reliability and separation quality.
Q: How is flow rate accuracy measured in HPLC?
Flow rate accuracy is checked to ensure the pump delivers the correct volume of mobile phase.
Method:
Collect HPLC-grade water in a calibrated volumetric flask for a known time using a stopwatch.
Calculation:
Acceptance criterion: Observed flow rate should be within ±2.0% of the set value.
This ensures accurate solvent delivery and consistent chromatographic performance.
Q: What is the acceptance criterion for flow rate consistency in HPLC?
Flow rate consistency ensures the pump delivers a stable and repeatable flow over time.
It is evaluated by multiple injections (typically six) of a standard solution like caffeine.
Parameter checked: %RSD (Relative Standard Deviation) of retention times.
Acceptance criterion: %RSD of retention time should be Not More Than (NMT) 1.0%.
A low %RSD confirms smooth pump operation and stable chromatographic performance.
Q: How is flow rate consistency evaluated in HPLC?
Flow rate consistency checks the stability of pump delivery over repeated runs.
Procedure: Inject a standard solution (commonly 10 ppm caffeine) six times under identical conditions.
Record the retention time for each injection.
Calculate the %RSD (Relative Standard Deviation) of the retention times.
Acceptance criterion: %RSD should be Not More Than 1.0%.
A low %RSD indicates stable pump performance and reliable flow delivery.
Q: What is meant by compositional accuracy in HPLC?
Compositional accuracy refers to the precision of gradient mixing between solvents in an HPLC system.
It ensures that the actual solvent ratio delivered by the pump matches the programmed composition.
Evaluated by: Running a test mixture (e.g., 0.25% acetone in water) and measuring absorbance at specific wavelengths.
Acceptance criterion: Observed composition should be within the specified tolerance (typically ±2%).
Accurate composition is critical for reproducible gradients, consistent retention times, and reliable separations.
Q: What is the delay volume limit for the pump system in HPLC?
Delay volume (or dwell volume) is the volume between the point of solvent mixing and the column inlet.
It affects how quickly gradient changes reach the column.
Measured by: Monitoring the change in detector response after switching mobile phases of different compositions.
Acceptance criterion: Delay volume should be Not More Than (NMT) 1.0 mL.
A low delay volume ensures faster gradient response, better reproducibility, and sharper separations.
Q: What is the acceptable range for injection volume accuracy in HPLC?
Injection volume accuracy ensures the autosampler delivers the correct sample volume each time.
It directly impacts peak area, quantification accuracy, and reproducibility.
Test: Perform 10 consecutive injections of a fixed volume (e.g., 20 µL).
Acceptance criterion: The average injected volume should be 20 ± 0.4 µL (i.e., within ±2%).
Accurate injection volume ensures consistent peak responses and reliable analytical results.
Q: How is injection volume accuracy determined in HPLC?
Injection volume accuracy verifies that the autosampler dispenses the programmed sample volume correctly.
Procedure:
Weigh an HPLC vial before and after 10 consecutive injections.
Calculate the difference in weight to determine the total injected volume (using solvent density).
Compute the average injection volume.
Acceptance criterion:
Mean injected volume should be within ±2% of the set value (e.g., 20 ± 0.4 µL).
Ensures precise sample delivery and consistent quantification in chromatographic analysis.
Q: What is the limit for injection volume precision in HPLC?
Injection volume precision ensures the autosampler delivers the same volume repeatedly with minimal variation.
Test: Perform multiple (usually six) injections of a standard solution under identical conditions.
Calculate the %RSD (Relative Standard Deviation) of peak areas or retention times.
Acceptance criterion: %RSD should be Not More Than (NMT) 1.0%.
Low %RSD confirms high repeatability, reliable autosampler performance, and consistent analytical results.
Q: How is injection volume linearity evaluated in HPLC?
Injection volume linearity checks the proportionality between injected volume and detector response.
Procedure: Inject a standard solution at varying volumes (e.g., 5, 10, 20, 50, and 100 µL).
Record the peak area for each injection.
Plot a graph of peak area vs. injection volume.
Calculate the correlation coefficient (R²).
Acceptance criterion: R² should be Not Less Than (NLT) 0.9990.
A high R² value confirms accurate sample delivery and linear detector response.
Q: What is the acceptance criterion for autosampler temperature in HPLC?
Autosampler temperature control ensures sample stability and consistency during analysis.
Test: Set the autosampler to a specific temperature and measure it using a calibrated thermometer or probe.
Acceptance criterion: The measured temperature should be within ±2°C of the set temperature.
Proper temperature control prevents sample degradation, evaporation, or variability in peak response.
Ensures reliable and reproducible chromatographic results.
Q: What are the required set temperatures for column oven calibration in HPLC?
Column oven calibration ensures accurate and uniform temperature control during chromatographic runs.
Purpose: To verify that the oven maintains correct temperatures across the working range.
Required set temperatures: 60°C, 50°C, 30°C, 20°C, and 10°C.
Temperature readings are compared using a calibrated thermometer or probe.
Accurate oven calibration ensures stable retention times, consistent separations, and reproducible results.
Q: What is the acceptance limit for column oven temperature accuracy in HPLC?
Column oven temperature accuracy ensures consistent thermal conditions for reproducible chromatography.
Test: Compare the set temperature with the actual temperature measured using a calibrated thermometer or probe.
Acceptance criterion: The measured temperature should be within ±2°C of the set value.
Accurate temperature control helps maintain stable retention times, peak shapes, and method reliability.
Q: How is the temperature measured in HPLC column oven calibration?
Temperature measurement ensures the column oven maintains the correct set temperature.
Procedure:
Place a calibrated digital thermometer or temperature probe inside the oven near the column position.
Allow the system to stabilize at each set temperature (e.g., 10°C to 60°C).
Record the actual temperature reading and compare it with the set value.
Acceptance criterion: Actual temperature should be within ±2°C of the set temperature.
This confirms accurate thermal control and ensures reliable chromatographic performance.
Q: How is wavelength accuracy evaluated in HPLC?
Wavelength accuracy ensures the detector operates at the correct wavelength for precise detection.
Using known UV maxima and minima, such as:
Maxima 273 ± 2 nm, Maxima 205 ± 2 nm, and Minima 245 ± 2 nm.
Procedure:
Use a reference standard with known UV absorbance peaks (e.g., holmium oxide or didymium filter).
Scan the spectrum and record observed absorbance maxima and minima.
Compare observed wavelengths with certified reference values.
Acceptance criterion: Observed peaks should be within ±2 nm of the standard reference wavelengths.
Accurate wavelength calibration ensures reliable detection, quantification, and spectral integrity.
Q: What is the acceptance criterion for detector linearity in HPLC?
Detector linearity verifies the proportional response of the detector to varying analyte concentrations.
Procedure: Inject standard solutions at multiple concentrations and record the corresponding peak areas.
Plot a calibration curve of concentration vs. peak area.
Acceptance criterion: The correlation coefficient (R²) should be Not Less Than (NLT) 0.9990.
High linearity confirms the detector’s accuracy, sensitivity, and suitability for quantitative analysis.
Q: What is the typical injection volume used for detector calibration in HPLC?
Detector calibration ensures accurate and consistent signal response at known sample volumes.
Typical injection volume: 10 µL of standard solution.
This volume provides an optimal balance between signal intensity and baseline stability.
Using a consistent injection volume ensures reliable calibration, reproducible peak areas, and accurate detector performance evaluation.
Q: What is the standard mobile phase composition for calibration in HPLC?
The standard mobile phase provides consistent elution and detector response during calibration.
Commonly used composition: Methanol : Water (50:50 v/v).
This balanced solvent system ensures moderate polarity, suitable for most UV-active compounds.
It offers stable baseline, good peak shape, and reproducible retention times.
A standardized mobile phase helps maintain method consistency and reliable calibration results.