Top 50 FAQs on HPLC For Beginners

It includes More than 50 Frequently asked Questions with answers on HPLC i.e. FAQs on HPLC for beginners :

(Frequently asked Questions) FAQs on HPLC – High Performance Liquid Chromatography for Beginners :

1. What is HPLC? High Performance Liquid Chromatography (HPLC) is an analytical technique used to separate and quantify components of a mixture.
2. What are the components of an HPLC system? An HPLC system basically includes a pump, an injector, a column, a Detector and Recorder.
3. What is the function of the pump in an HPLC system? The pump is responsible for delivering the mobile phase (solvent or buffer) at a constant flow rate and pressure through the column.
4. What is the function of the injector in an HPLC system? The injector introduces the sample into the mobile phase stream and onto the column for separation.
5. What is the function of the column in an HPLC system? The column is where the separation of the components of the mixture takes place.
6. What is the function of the detector in an HPLC system? The detector detects and quantifies the separated components
7. What are the types of columns used in HPLC? There are two types of columns used in HPLC: Packed columns and Capillary columns.
8. What are the types of detectors used in HPLC? There are several types of detectors used in HPLC, including UV-Vis, fluorescence, electrochemical, mass spectrometry, and refractive index detectors.
9. What is the difference between HPLC and GC? HPLC separates components based on their solubility in the mobile phase, while GC separates components based on their volatility.
10. What is the mobile phase in HPLC? The mobile phase in HPLC is the liquid solvent or buffer that is used to elute the components of the mixture through the column.
11. What is the stationary phase in HPLC? The stationary phase in HPLC is the solid or liquid phase that is immobilized within the column and interacts with the components of the mixture.
12. What is the difference between a normal phase column and a reverse phase column? In a normal phase column, the stationary phase is polar and the mobile phase is non-polar, while in a reverse phase column, the stationary phase is non-polar and the mobile phase is polar.
13. What is the difference between isocratic and gradient elution in HPLC? In isocratic elution, the mobile phase composition is constant throughout the separation, while in gradient elution, the mobile phase composition changes over time.
14. What is the retention time in HPLC? Retention time is the time it takes for a component of the mixture to travel through the column and reach the detector.
15. What is the peak in HPLC? A peak is a graphical representation of the amount of a component of the mixture that has reached the detector at a particular retention time.
16. What is the area under the curve in HPLC? The area under the curve is a measure of the amount of a component of the mixture that has been separated and detected by the HPLC system.
17. What is the resolution in HPLC? Resolution is a measure of the degree of separation between two adjacent peaks in the chromatogram.
18. What is the calibration curve in HPLC? A calibration curve is a plot of the peak area or height of a known concentration of standard solutions versus their respective concentrations.
19. What is the limit of detection (LOD) in HPLC? The LOD is the lowest concentration of a component that can be detected with a certain level of confidence.
20. What is the limit of quantification (LOQ) in HPLC ? The LOQ is the lowest concentration of a component that can be reliably quantified with a certain level of confidence.

21. What is the peak shape in HPLC? Peak shape refers to the shape of a chromatographic peak, which can be affected by various factors such as column performance, sample matrix, and injection volume.
22. What is peak asymmetry in HPLC? Peak asymmetry refers to the degree to which the tail of a chromatographic peak is distorted or skewed, which can be caused by column overload, injection volume, or column overpressure.
23. What is the dead volume in HPLC? The dead volume in HPLC is the volume of the mobile phase that is present between the injector and the column inlet, which can affect peak broadening and resolution.
24. What is the back pressure in HPLC? The back pressure in HPLC is the pressure exerted by the mobile phase as it flows through the column, which can be affected by factors such as column length, particle size, and flow rate.
25. What is the flow rate in HPLC? The flow rate in HPLC is the rate at which the mobile phase is pumped through the column, which can affect the speed and efficiency of the separation.
26. What is the injection volume in HPLC? The injection volume in HPLC is the volume of sample that is injected onto the column, which can affect peak shape, resolution, and sensitivity.
27. What is the sample preparation in HPLC? Sample preparation in HPLC involves various techniques such as filtration, extraction to prepare the sample for analysis.
28. What is the validation of an HPLC method? The validation of an HPLC method involves verifying the accuracy, precision, specificity, and robustness of the method to ensure that it produces reliable and reproducible results.
29. What is the difference between method development and method validation in HPLC? Method development involves selecting the appropriate column, mobile phase, and operating conditions to achieve the desired separation, while method validation involves verifying the performance of the developed method.
30. What is the role of pH in HPLC? The pH of the mobile phase can affect the ionization state and polarity of the components of the mixture, which can influence their retention and separation on the column.
31. What is the role of temperature in HPLC? Temperature can affect the viscosity, diffusivity, and solubility of the mobile phase and the sample components, which can influence the speed and efficiency of the separation.
32. What is the role of pressure in HPLC? Pressure can affect the flow rate, back pressure, and efficiency of the separation, which can influence the quality of the chromatogram.
33. What is the role of column length in HPLC? Column length can affect the resolution, retention, and sensitivity of the separation, but it can also increase the analysis time and consume more mobile phase.
34. What is the role of particle size in HPLC? Particle size can affect the surface area, porosity, and packing efficiency of the stationary phase, which can influence the separation efficiency and back pressure.
35. What is the role of column diameter in HPLC? Column diameter can affect the sample loading capacity, peak capacity, and resolution of the separation, but it can also influence the analysis time and consume more mobile phase.
36. What is the role of the detector wavelength in HPLC? Detector wavelength can affect the sensitivity and selectivity of the detector for specific components, as different components absorb light at different wavelengths.
37. What is the role of the mobile phase composition in HPLC? The mobile phase composition in HPLC can affect the retention, selectivity, and resolution of the separation, as different solvents and additives can influence the interaction between the sample components and the stationary phase.

38. What is the role of the stationary phase in HPLC? The stationary phase in HPLC is the solid support material that is coated with a liquid phase or chemically modified to selectively interact with the sample components, which can influence their retention and separation on the column.
39. What is the role of the guard column in HPLC? The guard column in HPLC is a short column that is placed before the analytical column to protect it from contamination or clogging, which can improve the longevity and reproducibility of the analytical column.
40. What is the role of the sample solvent in HPLC? The sample solvent in HPLC is the liquid in which the sample is dissolved or suspended before injection, which can affect the solubility, stability, and compatibility of the sample with the mobile and stationary phases.
41. What is the role of the injection solvent in HPLC? The injection solvent in HPLC is the liquid in which the sample is diluted or dissolved before injection, which can affect the compatibility and peak shape of the sample with the mobile and stationary phases.
42. What is the role of the sample matrix in HPLC? The sample matrix in HPLC refers to the complex mixture of components and impurities that are present in the sample, which can affect the selectivity, sensitivity, and accuracy of the analysis.
43. What is the role of the buffer system in HPLC? The buffer system in HPLC is a mixture of weak acid and its conjugate base or weak base and its conjugate acid that is added to the mobile phase to maintain a constant pH, which can improve the stability and reproducibility of the separation.
44. What is the role of the internal standard in HPLC? The internal standard in HPLC is a known amount of a compound that is added to the sample before analysis to monitor the sample preparation, injection, and detection processes, which can improve the accuracy and precision of the analysis.
45. What is the role of the gradient elution in HPLC? Gradient elution in HPLC is a technique in which the composition of the mobile phase is gradually changed during the separation to improve the resolution and selectivity of the separation.
46. What is the role of the isocratic elution in HPLC? Isocratic elution in HPLC is a technique in which the composition of the mobile phase is kept constant during the separation to achieve a simple and fast separation of the components.
47. What is the role of the solvent strength in HPLC? Solvent strength in HPLC refers to the ability of the mobile phase to dissolve or elute the sample components from the stationary phase, which can be controlled by adjusting the polarity, pH, and additives of the mobile phase.
48. What is the role of the column equilibration in HPLC? Column equilibration in HPLC is the process of stabilizing the column and the mobile phase at the initial conditions before the sample injection, which can improve the reproducibility and accuracy of the analysis.
49. What is the role of the rinse solvent in HPLC? The rinse solvent in HPLC is a solvent that is used to clean and condition the column after the analysis to remove any residual sample or impurities and restore the performance of the column.
50. What is the role of the detector response factor in HPLC? The detector response factor in HPLC is the ratio of the detector response for a given analyte to its concentration in the sample, which can be used to quantify the amount of the analyte in the sample based on the detector signal.
51. What is the role of the system suitability test in HPLC? The system suitability test in HPLC is a series of tests that are performed to evaluate the performance of the HPLC system before and during the analysis, which can ensure the reliability and accuracy of the analysis.
52. What is the role of the peak area in HPLC? The peak area in HPLC is the area under the chromatographic peak that represents the amount of the analyte that is separated and detected by the detector, which can be used to quantify the amount of the analyte in the sample.
53. What is the role of the peak height in HPLC? The peak height in HPLC is the height of the chromatographic peak that represents the intensity of the detector signal for the analyte, which can be used to estimate the amount of the analyte in the sample.
54. What is the role of the retention time in HPLC? The retention time in HPLC is the time that it takes for a sample component to elute from the column after injection, which can be used to identify and quantify the sample components based on their characteristic retention times.
55. What is the role of the theoretical plates in HPLC? The theoretical plates in HPLC are the imaginary stages of the column that represent the efficiency of the separation, which can be used to calculate the number of plates and the plate height of the column.
56. What is the role of the peak tailing in HPLC? The peak tailing in HPLC is the asymmetry of the chromatographic peak that can occur due to various factors such as sample matrix, column contamination, or flow rate, which can affect the accuracy and precision of the analysis.
57. What is the role of the peak fronting in HPLC? The peak fronting in HPLC is the asymmetry of the chromatographic peak that can occur due to factors such as overloaded column, high sample viscosity, or fast flow rate, which can affect the accuracy and precision of the analysis.
58. What is the difference between HPLC and UPLC? HPLC (High-Performance Liquid Chromatography) and UPLC (Ultra-Performance Liquid Chromatography) are both liquid chromatography techniques used for separating and analyzing compounds in a sample. The main difference between them is the particle size of the stationary phase. UPLC uses smaller particles (less than 2 μm), which allows for faster separations and higher resolution compared to HPLC.
59. What is the role of the pre-column filter in HPLC? The pre-column filter in HPLC is a small filter that is placed before the injector to remove particulate matter and other impurities from the sample before it enters the analytical column. The pre-column filter can protect the column and detector from damage and improve the reproducibility of the analysis.
60. What are the types of injectors used in HPLC? The types of injectors used in HPLC include manual injectors, automated injectors, and autosamplers.