lcms an instrument used for separation nd quantification

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PRESENTED BY: JEEJO N.U

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LC-MS
• LC/MS is a hyphenated technique, combining
the separation power of HPLC, with the
detection power of mass spectrometry.
• Here, an interface is 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
GAS PHASE  ELECTRON IMAPCT (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 & oligonucleotides having MW of
104 Da.
• Also used in the charactreization of 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
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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.
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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
polarity or 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
information and accurate quantitative analysis.
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17. APPLICATIONS
DRUG DEVELOPMENT:
• HPLC-MS is frequently used in drug development
at many different stages including:
Peptide Mapping
Glycoprotein Mapping
Natural Products counter screening.
Bioaffinity Screening
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18. PHARMACOKINETICS
 HPLC-MS is very commonly used in
the pharmacokinetic studies 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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