LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY 1
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LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
1 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.2Components of LCMS: 3 HPLCINTERFACEMASSANALYSERDETECTORExtraction of analyte from solvent.Ion evaporation and ionisation.Instrumentation of LC-MS: 1. INTERFACES: Moving Belt Interface.
ATMOSPHERIC PRESSURE IONIZATION: Electrospray Ionization.Atmospheric Pressure Chemical Ionization.Atmospheric Pressure Photoionization.
41. Moving Belt Interface5
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.
67ADVANTAGE: 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.
2. Thermospray Interface:8
2. Thermospray Interface:
STEPS:Nebulisation of the eluent from a heated transfer tube.
Ionisation of analytes.
910 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)
I. Electrospray Ionisation:11
High electrical potentialI. Electrospray ionisation:
STEPS:Analyte solution flow passes through electrospray needle having high potential difference.
Formation of charged droplets.
Gas phase ion formation.
1213ADVANTAGES:Study of higher molecular weight substances.Practical mass range up to 70000 DaAnalyze multiply charge compounds
DISADVANTAGES: Not applicable to non-polar and low polarity compounds. Salts and ion-pairing agents reduce sensibilityII. Atmospheric Pressure Chemical Ionization.14
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 0.2-2 ml/min.
1516ADVANTAGES: 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) III.Atmospheric Pressure Photoionization
17III. 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 (