1. 1 Principle  LC-MS is interfacing HPLC system with mass spectrometer.  The difficulty in hyphenation is to transform the solute into gas phase ion.  This problem is solved by using interfaces.  Hence the general principle involves the vaporization of the effluent and then ionization of the analyte which is further analyzed through the MS.

2. PROBLEMS IN COMBINING HPLC AND MS HPLC Liquid phase operation 25 - 50 deg. C No mass range limitations Inorganic buffers 1 ml/min eluent flow is equivalent to 500 ml/min of gas MS  Vacuum operation  200 - 300 deg. C  Up to 4000 Da for quadrupole MS  Requires volatile buffers  Accepts 10 ml/min gas flow

3. Components of LCMS: 3 INTERFACEDETECTOR Extraction of analyte from solvent. Ion evaporation and ionisation.

4. INSTRUMENTATION 1HPLC SYSTEM 2INTERFACES 3 MASS ANALYZERS 4 DETECTORS

5. 1. INTERFACES 1. Moving Belt Interface. 2.Thermospary Interface. 3.ATMOSPHERIC PRESSURE IONIZATION: I. Electrospray Ionization. II.Atmospheric Pressure Chemical Ionization. III.Atmospheric Pressure Photoionization.

6. 1. Moving Belt Interface 6

7. 1. Moving belt interface:  STEPS: I. Sample deposited onto a moving belt or wire. II. Sample passes through multiple vaccum zones. III. Sample is desorbed into source using heat. IV. Belt cycles back.  ADVANTAGE: Used with wide range of HPLC conditions, Flow rates and mobile phase.  DISADVANTAGES: Appearance of intense chemical background. Not suitable for thermally labile compounds. Surface effect reduces detection limits. 7

8. 2. Thermospray Interface: 8

9.  STEPS: I.Nebulisation of the eluent from a heated transfer tube. II.Ionisation of analytes.  ADVANTAGE: Easier to use at flow rate 2 ml\min.  DISADVANTAGES: Mobile phase should be volatile. Decomposition of thermally liable analytes. Not suitable for high molecular weight analyte.(>1000Da) Temperature optimization is critical. 9

10. I. Electrospray Ionisation: 10 High electrical potential

11. I. Electrospray ionisation:  STEPS: I.Analyte solution flow passes through electrospray needle having high potential difference. II.Formation of charged droplets. III.Gas phase ion formation.  ADVANTAGES: Applicable to volatile and non-volatile analytes. Study of higher molecular weight substances. Analyze multiply charged compounds. Practical mass range up to 70000 Da  DISADVANTAGES : Not applicable to non-polar and low polarity compounds. Salts and ion-pairing agents reduce sensibility. 11

12. II. Atmospheric Pressure Chemical Ionization. 12

13. II. Atmospheric Pressure Chemical Ionisation:  STEPS: I.HPLC effluent is passed through a pneumatic nebulizer where the droplets are both generated and desolvated. II.The spray so formed then passes through a heated region where the vapour is dried. III.Solvent molecules are ionised by corona discharge to generate stable reaction ions. IV.Liquid flow rate 0.2-2 ml/min.  ADVANTAGES: It is best applied to compounds with low to moderately high polarities.  DISADVANTAGES: Thermal degradation can occur. Limited structural information as multiple charging do not take place. Not appropriate for higher MW (e.g.,>1000 Da) and analyte that are charged in solution. 13

14. III.Atmospheric Pressure Photoionization 14

15. III. Atmospheric Pressure Photoionisation: STEPS: I.A vaporizer converts the LC eluent to the gas phase. II.A discharge lamp generates photons in a narrow range of ionization energies. ADVANTAGE OVER APCI: Applicable to highly non polar compounds and low flow rates (<100 μl/min). 15

16. 2. MASS ANALYSERS: 1.Magnetic Sector System. 2.Quadrapole Mass Analyser. 3.Ion Trap. 4.Time Of Flight. 16

17. 1. Magnetic Sector System 17 Flight Tube

18. 1. Magnetic Sector System: I.Ion optics in the ion-source chamber extract and accelerate ions. II.The ions enter the flight tube between the poles of a magnet and are deflected by the magnetic field. III.By varying the acceleration voltage or the magnetic field strength, a complete spectrum (an m/z range of e.g. 50 to 2000) can be obtained. IV.The accelerated ions of different masses were detected at different impact points on the detector plate and mass ratios are measured. 18

19. 2. Quadrapole Mass Analyser: F 19

20. 2. Quadrapole Mass Analyser:  It allows ions of any m/z ratio to pass through the metallic rods where a potential difference is applied and the instrument acts as a mass filter.  Quadrupole mass analyzers can operate in two modes: I. Scanning (scan) mode. II. Selected ion monitoring (SIM) mode.  A triple quadrupole tandem mass unit(ms-ms) has three quadrupoles arranged in a series. It can be set either for the analysis of intact peptides or their fragment ions.  Q1 is used to scan across a preset m/z range and select an ion of interest.  Q2 focuses and transmits the ions while introducing a collision gas into the flight path of the selected ion.  Q3 serves to analyze ion fragments. 20

21. 3. Ion Trap Mass Analyser: 21

22. 3.Ion Trap Mass Analyser: I.It consists of a circular ring electrode & two end caps that together form a chamber. II.Ions entering the chamber are trapped by electromagnetic field. Another field can be added to selectively eject ions from the trap. Mechanisam:  Destabilise the ions and eject them progressively from the trap.  Keep only one ion of a given m/z value in the trap and then eject it to observe it specifically.  Keep only one ion in the trap fragment it by inducing vibrations and observe the fragments. This is MS/MS.  The advantages of the ion-trap mass spectrometer include compact size and the ability to trap and accumulate specific ions. It carry out MS n i.e. multiple levels of fragmentation with out addition mass analyser. 22

23. 4. Time Of Flight. 23

24. 4. Time Of Flight: I. Uniform electromagnetic force is applied to all ions. II. Lighter ions travel faster & arrive at detector first.  After the initial acceleration phase, the velocity reached by an ion is inversely proportional to its mass.  These are commonly used in studies of proteins and protein fragments because this type of detector can handle and analyze very large molecular and fragmentation ions. 24

25. 3. Detectors: 1.Electron Multiplier Detector. 2.Dynolyte Photomultiplier Detector. 25

26. 1. Electron Multiplier Detector. 26

27. 1. Electron multiplier Detector:  A conversion dynode is used to convert either negative or positive ions into electrons.  These electrons are amplified by a cascade effect to produce a current. 27

28. 2. Dynolyte Photomultiplier Detector. 28 Photocathode

29. 2. Dynolyte Photomultiplier Detector:  Ions exiting the quadrupole are converted to electrons by a conversion dynode.  These electrons strike a phosphor which when excited,emit photons.  The photons strike a photocathode at the front of the photomultiplier to produce electrons and the signal is amplified by photomultiplier. 29

30. Applications Molecular Weight Determination Differentiation of similar octapeptides Determining the molecular weight of green fluorescent protein Structural Determination Structural determination of ginsenosides using MSn analysis Pharmaceutical Applications Rapid chromatography of benzodiazepines Detection of degradation products for salbutamol Identification of bile acid metabolites Biochemical Applications Rapid protein identification using capillary LC/MS/MS and database searching

31. Clinical Applications High-sensitivity detection of trimipramine and thioridazine Food Applications Identification of aflatoxins in food Determination of vitamin D3 in poultry feed supplements using MS3 Environmental Applications Detection of phenylurea herbicides Detection of low levels of carbaryl in food