1. LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

2. INTRODUCTION:
LC-MS is an hyphenated technique of analytical chemistry that
combines separation ability of HPLC with detection ability of
mass spectrometry.
It gives high sensitivity and selectivity.

3. Components of LCMS: HPLC INTERFACE MASS ANALYSER DETECTOR
Extraction of analyte from solvent.
Ion evaporation and ionisation.

4. Instrumentation of LC-MS:
1. INTERFACES:
Moving Belt Interface.
Thermospary Interface.
ATMOSPHERIC PRESSURE IONIZATION:
Electrospray Ionization.
Atmospheric Pressure Chemical Ionization.
Atmospheric Pressure Photoionization.

5. 1. Moving Belt Interface

6. 1. Moving belt interface:
STEPS:
Sample deposited onto a moving belt or wire.
Sample passes through multiple vaccum zones.
Sample is desorbed into source using heat.
Belt cycles back.

7. 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.

8. 2. Thermospray Interface:

9. 2. Thermospray Interface:
STEPS:
Nebulisation of the eluent from a heated transfer tube.
Ionisation of analytes.

10. Easier to use at flow rate 2 ml\min.
ADVANTAGE:
Easier to use at flow rate 2 ml\min.
DISADVANTAGES:
Mobile phase should be volatile.
Decomposition of thermally labile analytes.
Not suitable for high molecular weight analyte.(>1000Da)

11. I. Electrospray Ionisation:
High electrical potential

12. I. Electrospray ionisation:
STEPS:
Analyte solution flow passes through electrospray needle having high potential difference.
Formation of charged droplets.
Gas phase ion formation.

13. ADVANTAGES:
Study of higher molecular weight substances.
Practical mass range up to Da
Analyze multiply charge compounds
DISADVANTAGES:
Not applicable to non-polar and low polarity compounds.
Salts and ion-pairing agents reduce sensibility

14. II. Atmospheric Pressure Chemical Ionization.

15. II. Atmospheric Pressure Chemical Ionisation:
STEPS:
HPLC effluent is passed through a pneumatic nebulizer where the droplets are both generated and desolvated.
The spray so formed then passes through a heated region where the vapour is dried.
Solvent molecules are ionised by corona discharge to generate stable reaction ions.
Liquid flow rate ml/min.

16. 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)

17. III.Atmospheric Pressure Photoionization

18. III. Atmospheric Pressure Photoionisation:
STEPS:
A vaporizer converts the LC eluent to the gas phase.
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).

19. Magnetic Sector System. Quadrapole Mass Analyser. Time Of Flight.
2. MASS ANALYSERS:
Magnetic Sector System.
Quadrapole Mass Analyser.
Time Of Flight.

20. 1. Magnetic Sector System
Flight Tube

21. 1. Magnetic Sector System:
Ion optics in the ion-source chamber extract and accelerate ions.
The ions enter the flight tube between the poles of a magnet and are deflected by the magnetic field.
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.
The accelerated ions of different masses were detected at different impact points on the detector plate and mass ratios are measured . 

22. 2. Quadrapole Mass Analyser:F

23. 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:
Scanning (scan) mode.
Selected ion monitoring (SIM) mode.

24. 3. Time Of Flight.

25. Lighter ions travel faster & arrive at detector first.
4. Time Of Flight:
Uniform electromagnetic force is applied to all ions.
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.

26. Electron Multiplier Detector. Dynolyte Photomultiplier Detector.
3. Detectors:
Electron Multiplier Detector.
Dynolyte Photomultiplier Detector.

27. 1. Electron Multiplier Detector.

28. 1. Electron multiplier Detector:
A conversion dynode is used to convert ions into
electrons.
These electrons are amplified by a cascade
effect to produce a current.

29. 2. Dynolyte Photomultiplier Detector.
Photocathode

30. 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.

31. Applications Of LCMS: Molecular Weight Determination.
Structural Determination.
Pharmaceutical Applications.
Clinical Applications.
Food Applications.
Environmental Applications.

32. Molecular Weight Determination:
Example:
To determine molecular weight of n-butane.

33. Structural Determination.
Example:
1. Structural determination of ethyl sec-butyl ether.

34. Pharmaceutical Applications:
Example:
Identification and quantification of individual
benzodiazepines from an incompletely resolved mixture.

35. Food Applications: Example: 1.Identification of aflatoxins in food.
Clinical Applications: Example: High-sensitivity detection of trimipramine and thioridazine.
Food Applications:
Example:
1.Identification of aflatoxins in food.

36. Environmental Applications:
Example:
1.Detection of phenylurea herbicides.

37. Problems in combining HPLC & MS
Liquid phase operation.
25-50 °C.
1 ml/min eluent flow is equivalent to 500 ml/lit of gas. MS
Vaccum operation.
°C.
Accepts 10 ml/min gas flow.

38. REFERENCES: Willard Merritt Dean Settle, Instrumental Method
of Analysis Pg no.608.
Skoog, Holler & Crouch, Instrumental Analysis,
Pg no.606.

39. THANK YOU !!!