LC-MS-JEEJO.ppt

lcms an instrument used for separation nd quantification

1. 1PRESENTED BY: JEEJO N.U

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LC-MS
•LC/MS is a hyphenatedtechnique, combining
the separationpower of HPLC, with the
detectionpower of mass spectrometry.
•Here, an interfaceis needed which will eliminate
the solvent and generate gas phase ions.

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4. ION SOURCES FOR MOLECULAR MASS
SPECTROSCOPY
BASIC TYPE NAME
GASPHASE ELECTRONIMAPCT (EI)
CHEMICAL IONIZATION (CI)
FIELD IONIZATION (FI)
DESORPTION FIELD DESORPTION
ELECTROSPRAY IONIZATION (ESI)
MATRIX-ASSISTED DESORPTION-
IONIZATION (MALDI)
PLASMA DESORPTION (PD)
FAST ATOM BOMBARDMENT (FAB)
SECONDARY-ION MASS
SPECTROMETRY (SIMS)
THERMOSPRAY IONIZATION (TS)
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5. ELECTRON IMAPCT IONIZATION
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M + e
-
M
.+
+ 2e
-

6. ELECTROSPRAY IONIZATION (ESI)
•Electrospray is a method of getting the solution
phase ions into the gas phase so that they can
be sampled by the mass spectrometer.
•One of the most important techniques for
analyzing biomolecules such as proteins,
polypeptides & oligonucleotideshaving MW of
10
4
Da.
•Also used in the charactreizationof inorganic
species & synthetic polymers.
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7. Three Fundamental Processes:
1.Production of charged droplets.
2.Droplet size reduction, and fission.
3.Gas phase ion formation.
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8. ELECTROSPRAY
8+
+
+
+
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+
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+
++
+
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+
+
++
+
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+++
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To MS
Orifice
Plate
Curtain
Plate
Drying Gas
Curtain Gas
Droplet Formation
Desolvation
& Fission
Gas Phase Ion
Generation
5kV
Nebulizing
Gas

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ELECTROSPRAY IONISATION (ESI)
ELECTROSPRAY IONISATION (ESI )

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11. Droplet size reduction & fission
•Droplet size reduction occurs by the
continual repetition of two processes:
1.Desolvation(evaporation of neutral solvent and
volatile buffers).
2.Droplet fission caused by electric repulsion
between like charges.
11++
+
+
+
++++
+
+
+
+
+
+ +
+
+
+
+
+
+
++
+
+
+
+
+
+ ++
+
+
+
++++
+
+
+
+
+
+
++
+
+
+
++++
+
+
+
+
+
+
+
+
+
+
+
++++
+
+
+
+
+
+ +
+
+
+
+

12. Gas Phase Ion Formation
•MODELS:
•Charge Residue Model
•All the solvent evaporates, leaving a bare gas phase
ion.
•Ion Evaporation Model
•As the droplet shrinks the charges (analyte) that
reside on the surface get just enough energy to jump
into the gas phase.
•Ion Emission Model
•The high voltage cause some ion formation directly
from the LC capillary.
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13. ADVANTAGES
•Soft ionization technique, resulting in little
decomposition of labile analytes.
•Generally produces only molecular ions.
•Multi charged analytes easily produced,
allowing proteins to be analyzed.
•Wide range of analytes.
•Highly efficient ion production.
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14. MALDI-TOF MASS SPECTROSCOPY
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Sample (e.g.protein)
embedded in matrix is ionised
by laser.
Ions accelerated through
electric field at different rates.
Lightest arrive first.
For protein sequencing.

15. FAST ATOM BOMBARDMENT
•Used in the production of ions of polar high
molecular-mass species.
•Sample(condensed state) bombardment
with xenon or argon atoms.
•+ve & -ve analyte ions are sputtered from the
sample.
•Rapid sample heating reduces sample
fragmentation.
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16. •The primary advantage HPLC/MS has over GC/MS is
that it is capable of analyzing a much wider range of
components.
•Compounds that are thermally labile, exhibit high
polarityor have a high molecular mass may all be
analyzed using HPLC/MS.
•Lc-ms technique provide finger printing of the
particular elute.
•The combination can give molecular mass,structural
informationand accurate quantitative analysis.
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17. APPLICATIONS
DRUG DEVELOPMENT:
•HPLC-MS is frequently used in drug development
at many different stages including:
PeptideMapping
GlycoproteinMapping
NaturalProductscounterscreening.
BioaffinityScreening
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18. PHARMACOKINETICS
HPLC-MS is very commonly used in
the pharmacokineticstudies of
pharmaceuticals and is thus the most
frequently used technique in the field of
bio analysis.
These studies give information about how
quickly a drug will be cleared from the
hepatic blood flow, and organs of the body.
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19. REFERENCES
•Instrumental methods of analysis by
skoog,holler & crouch, page no:606-620.
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