BASIC COMPONENTS OF HPLCMS

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BASIC COMPONENTS OF HPLC-MS
MASS ANALSER
HPLC COLUMN
HPLC COLUMN
ION SOURCE
ION DETECTOR

• electrospray quadrupole electron
• APCI ion trap multiplier
• APPI time of flight
• LC-separation ionisation mass spectrum measuremen
  t
• (measurement of abundance
• of mass)

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IONISATION

• Atmospheric pressure ionisation (API)
• There are several different types of ionisation
  e.g.
• electrospray ionisation (ESI)
• atmospheric pressure chemical ionisation (APCI)
• atmospheric pressure photoionisation (APPI)

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ELECTROSPRAY IONISATION (ESI)

• organic acids (formic, acetic) promote ionisation
  of basic analytes to MH
• adduct formation (as by-product) with alkali
  metals or ammonium ions
• recommended buffers should be volatile (ammonium
  formate or acetate) 2-10 mM
• Ionisation is very pH dependent, for charging the
  molecule

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ATMOSPHERIC PRESSURE CHEMICAL IONISATION

• discharge needle at high voltage ionises
  nebulizing gas (air or nitrogen) forming primary
  ions
• primary ions react immediately with solvent
  molecules to form reagent ions (H3O, CH3OH2)
• reagent ions react (by proton transfer) with
  analyte molecules forming MH in positive mode
  or M-H- in negative ion mode

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ATMOSPHERIC PRESSURE PHOTOSPRAY IONISATION SOURCE

• Uses UV lamp for ionisation (10eV)
• Uses toluene as dopant (ionised by UV to form
  photoions), co-vaporized
• Evaporation in a heated probe, similar to APCI
• Ion-molecule reactions initiated by the photoions
• Proton transfer analytes ionised as MH
• Charge transfer analytes ionised as M

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MALDI-TOF MASS SPEC.

• Matrix assisted laser desorption-ionization
  (MALDI), time-of-flight (ToF) , MS
• 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