LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY 1.

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LIQUID CHROMATOGRAPHY-MASS SPECTROMETR 1

Transcript of LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY 1.

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LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

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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.

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Components of LCMS:

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HPLC INTERFACEMASS

ANALYSERDETECTOR

Extraction of analyte from solvent.

Ion evaporation and ionisation.

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Instrumentation of LC-MS:

1. INTERFACES:

1. Moving Belt Interface.

2. Thermospary Interface.

3. ATMOSPHERIC PRESSURE IONIZATION:

I. Electrospray Ionization.

II. Atmospheric Pressure Chemical Ionization.

III. Atmospheric Pressure Photoionization.

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1. Moving Belt Interface

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MSBelt

fan From LC

Heater tip Clean-upheater

pump pump wiperexit

Solvent evaporation

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1. Moving belt interface:

STEPS:

I. Sample deposited onto a moving belt or wire.

II. Sample passes through multiple vaccum zones.

III. Sample is desorbed into source using heat.

IV. Belt cycles back.

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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.

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2. Thermospray Interface:

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2. Thermospray Interface:

STEPS:

I. Nebulisation of the eluent from a heated transfer tube.

II. Ionisation of analytes.

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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)

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I. Electrospray Ionisation:

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High electrical potential

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I. Electrospray ionisation:

STEPS:

I. Analyte solution flow passes through electrospray needle having high potential difference.

II. Formation of charged droplets.

III. Gas phase ion formation.

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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

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II. Atmospheric Pressure Chemical Ionization.

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II. Atmospheric Pressure Chemical Ionisation:

STEPS:I. HPLC effluent is passed through a pneumatic nebulizer

where the droplets are both generated and desolvated.

II. The spray so formed then passes through a heated region where the vapour is dried.

III. Solvent molecules are ionised by corona discharge to generate stable reaction ions.

IV. Liquid flow rate 0.2-2 ml/min.

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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)

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III.Atmospheric Pressure Photoionization

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III. Atmospheric Pressure Photoionisation:

STEPS:

I. A vaporizer converts the LC eluent to the gas phase.

II. 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).

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2. MASS ANALYSERS:

1. Magnetic Sector System.

2. Quadrapole Mass Analyser.

3. Time Of Flight.

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1. Magnetic Sector System

Flight Tube

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1. Magnetic Sector System:

I. Ion optics in the ion-source chamber extract and accelerate ions.

II. The ions enter the flight tube between the poles of a magnet and are deflected by the magnetic field.

III. 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.

IV. The accelerated ions of different masses were detected at different impact points on the detector plate and mass ratios are measured . 

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2. Quadrapole Mass Analyser:

F

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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:

I. Scanning (scan) mode.

II. Selected ion monitoring (SIM) mode.

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3. Time Of Flight.

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4. Time Of Flight:

I. Uniform electromagnetic force is applied to all ions.

II. 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. 25

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3. Detectors:

1. Electron Multiplier Detector.

2. Dynolyte Photomultiplier Detector.

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1. Electron Multiplier Detector.

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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.

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2. Dynolyte Photomultiplier Detector.

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Photocathode

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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.

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Applications Of LCMS:

1. Molecular Weight Determination.

2. Structural Determination.

3. Pharmaceutical Applications.

4. Clinical Applications.

5. Food Applications.

6. Environmental Applications.

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Molecular Weight Determination:

Example:

1. To determine molecular weight of n-butane.

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Structural Determination.Example:

1. Structural determination of ethyl sec-butyl ether.

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Pharmaceutical Applications:

Example:

1. Identification and quantification of individual

benzodiazepines from an incompletely resolved mixture.

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Clinical Applications:

Example: 1. High-sensitivity detection of trimipramine and thioridazine.

Food Applications:

Example:

1.Identification of aflatoxins in food.

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Environmental Applications:

Example: 1.Detection of phenylurea herbicides.

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Problems in combining HPLC & MS

HPLCLiquid phase operation.25-50 °C.1 ml/min eluent flow is

equivalent to 500 ml/lit of gas.

MSVaccum operation.200-300 °C.Accepts 10 ml/min gas

flow.

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REFERENCES:

1. Willard Merritt Dean Settle, Instrumental Method

of Analysis Pg no.608.

2. Skoog, Holler & Crouch, Instrumental Analysis,

Pg no.606.

3. www.agilent.com

4. www.Wikipidia.com

5. www.sciencedirect.com

6. www.molecularsciences.org

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THANK YOU !!!