Lc ms-salu ppt

liquid chromatography

1. LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY (LC-MS) Presentation by Salini.c First M.Pharm GraceCollege Of Pharmacy

2. Liquid chromatography–Mass spectrometry Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an ADVANCED ANALYTICAL INSTRUMNTAL technique that combines the physical separation capabilities of LIQUID CHROMATOGRAPHY (or HPLC) with the mass analysis capabilities of MASS SPECTROMETER VIGNAN PHARMACY COLLEGE 2

3. AIMS To identify the different proteins, peptides, drugs, in various samples also to study the bioequivalence in future. 3

4. ADVANTAGES OF LC-MS Provides compound identity. Provides sensitive response to most analytes . Provides compound class information. Provides compound structure. Provides sequence information. Provides molecular weight information. Provides the five 5s Speed Selectivity Specificity Sensitivity $...Low Cost per Information Content. 4

5. SAMPLE CONSIDERATION FOR LC-MS The analyte must have ionizable groups such as Amines, Carboxylic Acids, Ketones and Aldehydes. For best sensitivity, work at a pH where the analyte is ionized. i.e. for acid, Neutral to basic pH (7-9) and Acidic pH (3-4) for bases` 5

6. PARTS OF LC-MS Two key components in this process are the ion source, which generates the ions, and the mass analyzer, which sorts the ions. Several different types of ion sources are commonly used for LC/MS.

7. Sample Column Detector Eluent Port collector Ionization Source Mass analyzer Vacuum Detector Read out device 7 FLOW CHART OF LC-MS

8. Interface:- It is difficult to interface a liquid chromatography to a mass-spectrometer cause of the necessity to remove the solvent. The commnly used interface are:- (1) Thermo spray ionization (2) Moving belt interface coupling (3) Particle beam interface coupling (4) Continues flow FAB coupling (5)Electro/ion spray interface

9. 1 .Thermospray Interface- As the name thermospray implies, heating the liquid flow leaving an LC system creates a spray of superheated mist containing small liquid droplets. 9 The most successful method involves directing the liquid flow through an electrically heated capillary, which can be directly introduced into the MS ion source. The droplets are further vaporized as they collide against the walls of the heated ion source .

10. 2. Moving belt/wire interface- 10 The moving-belt interface separates the condensed liquid-phase side of the LC from the high vacuum of the MS and uses a belt to transport the analytes from one to the other. The mobile phase of the LC is deposited on a band and evaporated. The analytes remain on the continuously cycling belt and are transported from atmospheric pressure into the vacuum of the ion source through two differentially pumped vacuum locks. A heater in the ion source evaporates the sample from the belt allowing MS analysis.

11. 3. Particle Beam Interface (MAGIC)- MAGIC: monodisperse aerosol generation interface for chromatography . The LC eluent is forced through a small nebulizer using a He gas flow to form a stream of uniform droplets. These droplets move through a desolvation chamber and evaporate to a solid particle. These particles are separated from the gas and transported into the MS source using a differentially pumped momentum separator . 11

12. 4.Continues flow FAB coupling A 5-15µm/ min.liquid stream,mixed with 5% glycerol,thioglycerol or nitrobenzyl alcohol as FAB matrix,flows through a narrow bore fused silica capillary towards either a stainless steal frit or a (gold plated)FAB target. At the target or frit,a uniform liquid film is formed due to a suitable balance b/w solvent evaporation &solvent delivery. Ions are generated by bombardment of liquid film by fast atom or ions,common to FAB. The fast atom like Ar / Xe is used for bombarding the sample &ions are splittered out of the sol.&enters in to MC.

13. 5.Electron spray ionization. Generate analyte ions in solution before the analyte reaches the mass spectrometer. The LC eluent is sprayed ( nebulized ) into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes further dissociation of the analyte molecules. The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases. Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase. These ions are attracted to and pass through a capillary sampling orifice into the mass analyzer.

14. Mass Analyser :- They deflects ions down a curved tubes in a magnetic fields based on their kinetic energy determined by the mass, charge and velocity. The magnetic field is scanned to measure different ions. Types of mass analyzer:- (1) Quadrupole mass analyser (2) Time of flight (3) Single focusing mass analyser (4) Double focusing mass analyser

15. DETECTORS Farady cup Electron multiplier detector Continous dyanode electron multiplier Microchannel plates

16. APPLICATIONS : LC-MS USED IN FOLLOWING AREAS Drug Discovery. Clinical Analysis. Proteomics. Forensic Chemistry Drug Metabolism study. Environmental chemistry. Diagnostic studies. 16

17. REFERENCES : Text book of mass spectroscopy by James Barker-2 nd edittion pg:320-327 www.chm.bris.ac.uk/ms/theory/esi-ionisation. www.opdac.com/lc/contents/api.htm www.lcms.com www.wikipedia.com 17

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