LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

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.

Components of LCMS: HPLC INTERFACE MASS ANALYSER DETECTOR Extraction of analyte from solvent. Ion evaporation and ionisation.

Instrumentation of LC-MS: 1. INTERFACES: Moving Belt Interface. Thermospary Interface. ATMOSPHERIC PRESSURE IONIZATION: Electrospray Ionization. Atmospheric Pressure Chemical Ionization. Atmospheric Pressure Photoionization.

1. Moving Belt Interface

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.

ADVANTAGE: 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:

2. Thermospray Interface: STEPS: Nebulisation of the eluent from a heated transfer tube. Ionisation of analytes.

Easier to use at flow rate 2 ml\min. 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: High electrical potential

I. Electrospray ionisation: STEPS: Analyte solution flow passes through electrospray needle having high potential difference. Formation of charged droplets. Gas phase ion formation.

ADVANTAGES: Study of higher molecular weight substances. Practical mass range up to 70000 Da Analyze multiply charge compounds DISADVANTAGES: Not applicable to non-polar and low polarity compounds. Salts and ion-pairing agents reduce sensibility

II. Atmospheric Pressure Chemical Ionization.

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.

ADVANTAGES: 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

III. 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 (<100 μl/min).

Magnetic Sector System. Quadrapole Mass Analyser. Time Of Flight. 2. MASS ANALYSERS: Magnetic Sector System. Quadrapole Mass Analyser. Time Of Flight.

1. Magnetic Sector System Flight Tube

1. Magnetic Sector System: Ion optics in the ion-source chamber extract and accelerate ions. The ions enter the flight tube between the poles of a magnet and are deflected by the magnetic field. By varying the acceleration voltage or the magnetic field strength, a complete spectrum (an m/z range of e.g. 50 to 2000) can be obtained. The accelerated ions of different masses were detected at different impact points on the detector plate and mass ratios are measured . 

2. Quadrapole Mass Analyser: F

2. Quadrapole Mass Analyser: It allows ions of any m/z ratio to pass through the metallic rods where a potential difference is applied and the instrument acts as a mass filter. Quadrupole mass analyzers can operate in two modes: Scanning (scan) mode. Selected ion monitoring (SIM) mode.

3. Time Of Flight.

Lighter ions travel faster & arrive at detector first. 4. Time Of Flight: Uniform electromagnetic force is applied to all ions. Lighter ions travel faster & arrive at detector first. After the initial acceleration phase, the velocity reached by an ion is inversely proportional to its mass. These are commonly used in studies of proteins and protein fragments because this type of detector can handle and analyze very large molecular and fragmentation ions.

Electron Multiplier Detector. Dynolyte Photomultiplier Detector. 3. Detectors: Electron Multiplier Detector. Dynolyte Photomultiplier Detector.

1. Electron Multiplier Detector.

1. Electron multiplier Detector: A conversion dynode is used to convert ions into electrons. These electrons are amplified by a cascade effect to produce a current.

2. Dynolyte Photomultiplier Detector. Photocathode

2. Dynolyte Photomultiplier Detector: Ions exiting the quadrupole are converted to electrons by a conversion dynode. These electrons strike a phosphor which when excited,emit photons. The photons strike a photocathode at the front of the photomultiplier to produce electrons and the signal is amplified by photomultiplier.

Applications Of LCMS: Molecular Weight Determination. Structural Determination. Pharmaceutical Applications. Clinical Applications. Food Applications. Environmental Applications.

Molecular Weight Determination: Example: To determine molecular weight of n-butane.

Structural Determination. Example: 1. Structural determination of ethyl sec-butyl ether.

Pharmaceutical Applications: Example: Identification and quantification of individual benzodiazepines from an incompletely resolved mixture.

Food Applications: Example: 1.Identification of aflatoxins in food. Clinical Applications: Example: 1. High-sensitivity detection of trimipramine and thioridazine. Food Applications: Example: 1.Identification of aflatoxins in food.

Environmental Applications: Example: 1.Detection of phenylurea herbicides.

Problems in combining HPLC & MS Liquid phase operation. 25-50 °C. 1 ml/min eluent flow is equivalent to 500 ml/lit of gas. MS Vaccum operation. 200-300 °C. Accepts 10 ml/min gas flow.

REFERENCES: Willard Merritt Dean Settle, Instrumental Method of Analysis Pg no.608. Skoog, Holler & Crouch, Instrumental Analysis, Pg no.606. www.agilent.com www.Wikipidia.com www.sciencedirect.com www.molecularsciences.org

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