Liquid chromatography and mass spectrometry ppt.

LC -MS SYSTEM HISTORY AND MECHANISM OF ACTION

1. LC-MS/MS

2. DEFINITION Liquid Chromatography-Mass Spectrometry (LC-MS)  is a powerful analytical technique used to separate, identify, and quantify compounds in a sample

3. TWO COMPONENT Liquid Chromatography (LC):  A technique used to separate mixtures of compounds based on their interactions with a stationary phase (typically a column) and a mobile phase (solvent). Compounds are separated based on their polarity, molecular weight, and other chemical properties. Mass Spectrometry (MS):  A technique used to identify and quantify compounds by measuring their mass-to-charge ratio (m/z). It ionizes chemical species and sorts the ions based on their mass-to-charge ratio using electric and magnetic fields.

4. Applications of LC-MS:

5. Early History (1960s - 1980s) The Conceptual Beginnings:

6. 1970s - Early Interfaces and Prototypes : early 1970s, efforts were made to interface LC with MS. The main challenge was the removal of the large volume of solvent used in LC before the analyte could enter the high-vacuum environment of the mass spectromet Moving-Belt Interface (MBI)  was one of the first successful attempts to couple LC with MS. Developed by McFadden et al. in 1977 and commercialized by Finnigan, the MBI allowed LC effluent to be deposited on a moving belt, where solvents were evaporated, and analytes were introduced into the MS. However, it was mechanically complex and not widely adopted.

7. 1980s - New Interfaces and Improved Sensitivity Direct Liquid Introduction (DLI) Interface  was developed in the early 1980s. This interface directed a small portion of the LC flow into the MS, but it suffered from clogging issues. Thermospray Interface (TSP) , developed in 1983 by Vestal and co-workers, revolutionized LC-MS by providing better sensitivity and a more reliable way to introduce liquid samples. It allowed direct ionization of analytes from the liquid phase without needing extensive solvent evaporation, and it gained popularity for its ability to handle a wide range of compounds. Particle Beam Interfaces (PBI)- another alternative for coupling LC with MS.

8. Rapid Advancements (1990s) 1990s - Introduction of Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI): I ntroduction of  Electrospray Ionization (ESI)  by John Fenn in the late 1980s (who later won the Nobel Prize in Chemistry in 2002 for this work) was a turning point for LC-MS. ESI allowed for the direct ionization of large biomolecules like proteins and peptides from the liquid phase into the gas phase at atmospheric pressure, significantly expanding the scope of LC-MS.

9. Quadrupole, Time-of-Flight (TOF), and Hybrid Mass Analyzers : The 1990s also saw significant advancements in mass analyzers , with the development of  Quadrupole ,  Time-of-Flight (TOF) , and hybrid instruments like  Quadrupole-TOF (Q-TOF)  and  Triple Quadrupole ( QqQ )  MS systems. These advancements allowed for higher sensitivity, resolution, and mass accuracy, which were crucial for applications like proteomics, metabolomics, and drug development.

10. Modern Developments (2000s - Present) 2000s - Advances in Instrumentation and Software:

11. 2010s - Expanding Applications and High-Throughput Analysis:

12. 2020s - Emerging Technologies and Omics Applications:

13. Working Principle of LC-MS Liquid Chromatography (LC) Separation: Sample Introduction:  A liquid sample is injected into the  LC system , which consists of a high-pressure pump, a sample injector, a chromatographic column, and a detector. Chromatographic Column:  The sample passes through a column filled with a stationary phase (typically silica or polymer-based materials). Different components in the sample interact with the stationary phase and the mobile phase (solvent) to varying degrees.

14. Interface Between LC and MS: The interface is crucial for transferring the liquid effluent from the LC to the MS while removing the solvent to create ions suitable for MS analysis. Common interfaces include  Electrospray Ionization (ESI) ,  Atmospheric Pressure Chemical Ionization (APCI) , and  Atmospheric Pressure Photoionization (APPI) .

15. Ionization Process:  The interface converts the liquid phase analytes into gaseous ions. ESI, for example, involves applying a high voltage to the liquid to create a fine spray of charged droplets. As the solvent evaporates, the charged droplets decrease in size until they release ions into the gas phase.

16. Mass Spectrometry (MS) Detection:

17. Ion Detection:  After separation, ions are detected by a detector, which measures the abundance of ions at each m/z value. The output is a  mass spectrum  showing the relative abundance of detected ions as a function of their m/z.

18. Data Analysis: The resulting data is processed to generate a  chromatogram  and a  mass spectrum  for each compound detected. The chromatogram shows the separation of the components over time, while the mass spectrum provides information on the molecular weight and structure of the compounds.

19. Identification and Quantification:  Compounds can be identified by comparing their mass spectra with reference spectra or databases. Quantification is done by measuring the area under the peaks in the chromatogram.

20. Types of Interfaces in LC-MS The interface between LC and MS plays a key role in the system's functionality. Here are some commonly used interfaces: Electrospray Ionization (ESI):  Suitable for polar, non-volatile, and thermally labile compounds. It generates multiply charged ions, which is helpful for high-molecular-weight compounds like proteins and peptides.

21. Atmospheric Pressure Chemical Ionization (APCI):  Suitable for less polar and more volatile compounds. APCI operates at high temperatures and generates singly charged ions. Atmospheric Pressure Photoionization (APPI):  Useful for non-polar compounds that are difficult to ionize by ESI or APCI. APPI uses a UV lamp to ionize the analytes.

22. Advantages of LC-MS: High Sensitivity and Specificity:  Detects and quantifies compounds at very low concentrations. Versatility:  Capable of analyzing a wide range of compounds, including small molecules, peptides, and large biomolecules. Structural Information:  Provides valuable data on molecular weight, fragmentation patterns, and structural elucidation.

23. LC-MS INSTRUMENT

24. DIAGRAM OF LC-MS LC SYSTEM- SOLVENT RESERVIOR PUMP SAMPLE INJECTOR COLOUMN DETECTOR (OPTIONAL)

25. INTERFACE BETWEEN LC and MS

26. MASS SPECTROMETRY

27. DATA SYSTEM

28. FLOW CHART REPRESNTATION

29. CHROMATOGRA, MASS SPECTRUM, PEAK IDENTIFICATION QUANTIFICATION, COMPOUND IDENTIFICATION

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