SEMINAR ON
HIGH PERFORMANCE 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:
INTERFACE
Extraction
of analyte
from
solvent.
DETECTOR
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:
Electrospray Ionization.
Atmospheric Pressure Chemical Ionization.
Atmospheric Pressure Photoionization.
I.
II.
III.
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1. Moving Belt Interface
Solvent evaporation
Belt
MS
pump
Heater tip
Clean-up
heater
From L C
fan
pump
wiper
exit
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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.
I.
II.
III.
IV.
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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:

I.
STEPS:
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:
High
electrical
potential
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I. Electrospray ionisation:

I.
STEPS:
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:
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.
I.
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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.
I.
II.
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).
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2. MASS ANALYSERS:
1.
2.
3.
Magnetic Sector System.
Quadrapole Mass Analyser.
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:
Scanning (scan) mode.
Selected ion monitoring (SIM) mode.
I.
II.
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3. Time Of Flight.
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4.
I.
II.
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.
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3. Detectors:
Electron Multiplier Detector.
2. Dynolyte Photomultiplier Detector.
1.
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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.
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.
2.
3.
4.
5.
6.
Molecular Weight Determination.
Structural Determination.
Pharmaceutical Applications.
Clinical Applications.
Food Applications.
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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Mass spectra showing identification of structurally similar
aflatoxins:
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Environmental Applications:
Example:
1.Detection of phenylurea herbicides.
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Problems in combining HPLC & MS
HPLC



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.
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REFERANCES:
1.
2.
3.
4.
5.
6.
Willard Merritt Dean Settle, Instrumental Method
of Analysis Pg no.608.
Skoog, Holler & Crouch, Instrumental Analysis,
India Edition Pg no.606.
www.agilent.com
www.Wikipidia.com
www.sciencedirect.com
www.molecularsciences.org
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