High-Performance Liquid Chromatography(HPLC)

High-Performance Liquid Chromatography(HPLC). HPLC is a column chromatography. HPLC is a Liquid-solid type of chromatography. Where liquid is a mobile phase and solid is a stationary phase. The mobile phase contains methanol plus water, usually as a solvent. In reverse phase HPLC, the mobile phase is polar in nature. The stationary phase is made up of solid components like silica. In reverse phase HPLC stationary phase is a nonpolar in nature. Instrumentation of HPLC contain solvent chamber having a mobile phase, pump, injector, the column having a a stationary phase, detector, recorder, and computer monitor showing the graphical representation.

1. High-Performance Liquid Chromatography(HPLC) DR. ANURAG KUMAR J unior resident -3 Dept. of Pharmacology & Therapeutics K ing George’s Medical U niversity Lucknow

2. Learning objectives Introduction: Chromatography Classification & Principle HPLC: Classification/Functional units Applications Limitations References

3. Chromatography Principle: laboratory technique for the separation of a mixture into its components due to the differing time taken for each component to travel through a stationary phase when carried through it by a mobile phase

4. Classification of chromatography On the basis of the mobile & stationary phase: Mobile phase- Liquid/Gas Stationary phase- Solid/Liquid Liquid: Liquid-solid chromatography( eg ; CC) Liquid-liquid chromatography ( eg ; PC) Gas: Gas-liquid chromatography Gas-solid chromatography

5. Classification …. 1. Liquid chromatography (test for pollution in water samples like lakes and rivers) 2. Gas chromatography (detect bombs and valuable in forensic investigations) 3. Thin-layer chromatography (used to check the purity of organic compounds such as the presence of insecticide or pesticide in foods) 4. Paper chromatography (uses a strip of paper in the stationary phase).

6. Principles of Column Chromatography based on Adsorption Partition Molecular sieving Ion-exchange Affinity

7. High-performance liquid chromatography (HPLC) Formerly known as high-pressure liquid chromatography Technique in analytical chemistry used to Separate Identify Quantify specific components in mixtures

8. HPLC

9. Advantages over low-pressure column liquid chromatography Greater sensitivity (various detectors can be employed) Improved resolution Speed Easy sample recovery (less eluent volume to remove) A wide variety of stationary phases

10. Classification of HPLC 1. Normal phase HPLC (effective method for separating phospholipid classes) 2. Reverse phase HPLC (the most common method used to separate compounds that have hydrophobic moieties) 3. Size-exclusion HPLC/molecular sieve chromatography (Used in large molecules/macromolecular complexes such as industrial polymers and proteins) 4. Ion-exchange HPLC (separates ions and polar molecules according to their ion exchanger.

11. Normal phase HPLC They are also known as normal-phase or absorption chromatography It has a polar stationary phase and a non-polar mobile phase Therefore, the stationary phase is usually silica, and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures The technique is used for water-sensitive compounds, geometric isomers, cis-trans isomers, class separations, and chiral compounds.

12. Reverse phase HPLC The stationary phase is nonpolar (hydrophobic), while the mobile phase is an aqueous, moderate-polar It works on the principle of hydrophobic interactions; hence more nonpolar the material will be retained for longer This technique is used for non-polar, polar, ionizable, and ionic molecules.

13. Size-exclusion HPLC/molecular sieve chromatography It is also known as gel permeation chromatography or gel filtration chromatography The column is filled with a material having precisely controlled pore sizes, and the particles are separated according to their molecular size Larger molecules are washed more rapidly than smaller molecules through the column.

14. Size-exclusion chromatography is also helpful in determining the tertiary and quaternary structure of proteins and amino acids It is also used for the determination of the molecular weight of polysaccharides

15. Ion-exchange HPLC In this type of chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase Same charged ions are excluded This technique is used in purifying water and Ligand Eg. Ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of carbohydrates and oligosaccharides, etc.

16. Functional units/Instrumentation of HPLC Solvent Reservoir/Mobile Phase Pump Sample Injector Columns/Stationary phase Detector Data Collection Devices/Recorder

17. Monitor Sample chamber Detector Solvent reservoir Column Pump

18. 1. Solvent Reservoir/Mobile Phase Should be easily available Moderately viscus Having different polarities Contains a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample

19. 2. Pump Aspirates the mobile phase from the solvent reservoir and forces it through the system’s column and detector Depending on several factors, including- column dimensions, the particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated.

20. 3. Sample Injector The injector can be a single injection or an automated injection system An injector for an HPLC system should provide an injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).

21. 4. Columns Columns are usually made of polished stainless steel, having 50-300 mm length, and an internal diameter between 2-5 mm They are commonly filled with a stationary phase with a 3–10 µm particle size Columns with less than 2 mm internal diameters are often called microbore columns Ideally, the temperature of the mobile phase and the column should be kept constant during an analysis.

22. 5. Detector Located at the end of the column, detects the analytes as they elute from the chromatographic column Detector generates an analog electrical signal proportional to the concentration of the analyte (elutes) from the column This signal can be based on various principles, such as UV absorbance, fluorescence, or conductivity Eg ; UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.

23. 6. Data Collection Device: Recorder Signals from the detector will be collected on chart recorders or electronic integrators that has the ability to process, store and reprocess the chromatographic data A/D Conversion: The A/D converter takes a continuous analog signal from the detector and converts it into a series of discrete digital values It essentially samples the signal at a specific rate (sampling frequency) and assigns a digital code representing the signal strength.

24. It doesn't directly interact with the sample but translates the information from the detector into a visual representation via computer The computer integrates the detector’s response to each component and places it into a chromatograph that is easy to read and interpret.

25. There are two main types of recorders used in HPLC 1. Chart Recorders (Traditional): Older technology where a pen was used to draw a trace on a moving paper chart The detector signal (often voltage) controls the pen's position, resulting in a graph having time (x-axis) and response (y-axis) Each graph peak represents the elution of individual components from the sample mixture

26. 2. Computer-Based Data Processors (Modern): These are now considered the standard one The detector signal is converted into a digital format and processed by a computer The software displays the chromatogram on a screen, often with features for peak integration, analysis, and data export

27. Samples could be run simultaneously in lanes side by side and results were able to be seen directly on the plate in the form of colored bands.

28. The computer-based data processor receives the digital signal (after conversion by the A/D converter) and displays the chromatogram The software automatically detects peaks and calculates their area and height for analysis

29. Interpretation of graph Quantitative Analysis: Peak area is the preferred method for quantitative analysis due to its proportionality to the amount of the component Qualitative Analysis or Screening: Peak height can be helpful for comparing relative amounts of a component between samples or for initial screening purposes

30. Applications of HPLC Drug Discovery and Development: HPLC plays a crucial role in analyzing drug candidates during the development process It helps to purify new drug candidates from reaction mixtures Identify and quantify impurities in drug formulations Determine the stability and degradation profile of drugs Analyze drug metabolites in biological samples

31. 2. Clinical Research and Diagnostics Biomarker Analysis: Measuring the levels of specific molecules in biological fluids that can indicate disease or treatment response Analysis of Biological Samples: HPLC is used to analyze various components in biological samples, such as: Proteins and peptides for disease diagnosis or biomarker studies Nucleic acids (DNA and RNA) for genetic analysis Metabolic products for studying metabolic pathways

32. 3. Environmental analysis Pollution Monitoring: HPLC is used to detect and quantify various pollutants in environmental samples, such as: Pesticides in water and soil Industrial pollutants in air and wastewater Microbial toxins in food and water

33. Other Applications: Petroleum Industry: Analyzing the composition of crude oil and petroleum products Material Science: Characterizing polymers and other materials Cosmetics Industry: Analyzing the components of cosmetic products Food and Chemical industry

34. RF value Distance traveled by a particular component RF= Distance traveled by the solvent

35. Limitations HPLC is much more costly and requires a large number of expensive organics HPLC may have low sensitivity for certain compounds, and some cannot even be detected as they are irreversibly adsorbed Complexity Volatile substances are much better to be separated by gas chromatography.

36. References Malviya, Rishabha & Bansal, Vvipin & Pal, Om & Sharma, Pramod. (2010). High-performance liquid chromatography: A short review. Journal of Global Pharma Technology. 2. 22-26.  https://laboratoryinfo.com/hplc Sarkar S, Srivastava V, Mohanty M. Postgraduate pharmacology. 1st ed.Paras Medical Publisher;2020

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