Liquid Chromatoghraphy / Mass Spectroscopy lcms SPECTROSCOPY

1. LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY
(LC/MS)
PRESENTED BY:
RANJEET KUMAR YADAV
M.PHARM 1ST YEAR
(PHARMACEUTICAL
CHEMISTRY)
GUIDED BY:
MRS. CHANDANA MAAJI
ASSISTANT PROFESSOR
NIET (PHARMACY INSTITUTE)
GR. NOIDA (UP)
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2. CONTENT
Introduction
Principle
Instrumentation
Application
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3. INTRODUCTION
It refers to the combination of liquid chromatographic
separation with mass spectrometric detection for non polar,
low volatility, thermally unstable compounds.
 LC differentiates compounds by their physico-chemical
properties and MS differentiates compounds by mass
(specifically their mass-to-charge ratio).
 The mass spectrometer acts not only as the "LC detector"
but, it provides the capability to identify the species
corresponding to each chromatographic peak through its
unique mass spectrum.
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4. High sensitivity of mass spectroscopy provides the information
for identification of compound or structural elucidation of
compounds
In the LC/MS we remove the detector from the column of LC fit
the column to the interface of MS
In the most cases the interface used in LC/MS are ionization
source
As the metabolites appear from the end of the column they enter
the mass detector , where the solvent is removed and the
metabolites are ionized
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5. PRINCIPLE
Thermospray method:
 The thermospray method depends on the thermal generation of a
spray and further separate heat treatment of that spray to yield
desolvated ions.
 The heat creates a supersonic, expanding aerosol jet that contains
a mist of fine droplets of solvent vapour and sample molecules., the
droplets vaporize.
 The excess vapor is pumped away by an added mechanical pump,
which is directly coupled to the ion source.
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6.  Ions of the sample molecules are formed in the spray either by
direct desorption or by chemical ionization when used with polar
mobile phases that contain appropriate buffers.
 A conventional electron beam is used to provide gas phase
reagent, ions for the chemical ionization of solute molecules.
The ions are led into a quadrupole or magnetic sector mass
spectrometer , to flow rates efluents up to 2ml/min are permissible
Monodisperse Aerosol generation interface:
The interface is configured in three sections:
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7. 1. Aerosol generator
2. Desolvation chamber
3. Two stage aerosol beam pressure reducer.
 In the aerosol generates the high pressure effluent from the
chromatographic column, passes through a small diameter
orifice to form a fine liquid jet. The jet breaks up under natural
forces to form uniform drops, which are immediately dispersed
with gas stream introduced at right angles to the liquid flow
direction
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8. The solvent evaporates in the desolvation chamber. The chamber
is maintained near room temperature by heating gently to replace
the latent heat of vaporization necessary for solvent evaporation.
 The two stages aerosol beam separator consists of two nozzle
and skimmer devices.
 These reduces the pressure from an initial value close to
atmospheric pressure in the desolvation chamber to a final value
close to the pressure in the ion source.
 The separator also allows solute particles to be preferentially
transferred through the system, while dispersion of the gas and
solvent vapor are pumped away.
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9. INSTRUMENTATION
 HPLC system
• Ion source: used in the
vaporization, ionization of
target Molecule.
• Mass analyser: used to
separate the gas phase
ions by mass to-charge
ratio (m/z).
• Ion detector: detection of
the mass separated.
• Data system
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10. Mobile Phase
The mobile phase is the solvent that moves the solute through out
column.
General requirements:
(1)Low cost, UV transparency, high purity
(2) Low viscosity, low toxicity, non flammability.
(3) Non corrosive to LC system component
Solvent strength and selectivity:
It is the ability of solvent to elute solutes from a column.
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11. COLUMN
The use of di-functional or tri-functional silanes to create
bonded groups with two or three attachment points leading to
phases with higher stability in low or higher pH and lower
bleed for LCMS
Most widely used columns for LC-MS are:
(1)fast LC column
the use of short column. (15-50mm)
(2) Micro LC column.
the use of large column. (20-150mm)
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12. Sample preparation
Sample preparation Sample preparation generally consists of
concentrating the analyte and removing compounds that can cause
background ion or suppress ionization.
Example of sample preparation include:
1. On Column concentration to increase analyte concentration.
2. Desalting to reduce the sodium and potassium adduct formation
that commonly occurs in electro spray.
3. Filtration to separate a low molecular-weight drug from
proteins in plasma, milk, or tissue.
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13. INTERFACES
LC-MS systems include a device for introducing samples (such as
an HPLC )an interface for connecting such device, an ion source
that ionizes samples, an electrostatic lens that efficiently introduces
the generated ions, a mass analyzer unit that separates ions based
on their mass-to-charge (m/z) ratio, and a detector unit that detects
the separated ions.
In an LC-MS system, however, if the LC unit is simply connected
directly to the MS unit, the liquid mobile phase would vaporize,
resulting in large amounts of gas being introduced into the MS unit.
This would decrease the vacuum level and prevent the target ions
from reaching the detector. So interfaces are to be used.
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14. TYPES OF INTERFACES
It is difficult to interface a liquid chromatography to a mass
spectrometer cause of the necessity to remove the solvent.
The commonly used interfaces are:
(1) Electrospray ionization (ESI)
(2) Thermospray ionization (TSI)
(3) Atmospheric pressure chemical ionization (APCI).
(4) Atmospheric pressure photoionization (APPI)
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15. 1. Electro Spray
lonization (ESI)
 ESI draws sample
solutions to the tip of a
capillary tube, where it
applies a high voltage
of about 3 to 5 kv.
 A nebulizer gas
flows from outside the
capillary to spray the
sample. This creates a
fine mist of charged
droplets with the same
polarity as the applied
voltage.
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16. As these charged particles move, the solvent continues to
evaporate, thereby increasing the electric field on the droplet
surface. When the mutual repulsive force of the charges
exceeds the liquid surface tension, then fission occurs
As this evaporation and fission cycle is repeated, the droplets
eventually become small enough that the sample ions are
liberated into the gas phase
ESI provides the softest ionization method available, which
means it can be used for highly polar, least volatile, or
thermally unstable compounds.
.
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17. 2. Atmospheric
pressure chemical
ionization (APCI)
APCI vaporizes
solvent and sample
molecules by spraying
the sample solution
into a heater (heated to
about 400 C) using a
gas, such as N2.
 Solvent molecules
are ionized by corona
discharge to generate
stable reaction ions.
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18. 3. Thermospray ionization (TSI)
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19. 4. Atmospheric
pressure
photoionization(APPI)
The LC eluent is
vaporized using a
heater at atmospheric
pressure.
 The resulting gas is
made to pass through a
beam of photons
generated by a
discharge lamp (UV
lamp) which ionizes
the gas molecules
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20. Mass Analyser
They deflections down a curved tubes in a magnetic fields based
on their kinetic energy determined by the mass, charge and
velocity.
The magnetic field is scanned to measure different ions.
 Types of mass analyzer:
(1)Quadrapole mass filter.
(2) Time of flight
(3) lon trap
(4) Fourier transform ion cyclotron resonance (FT-ICR) 20
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21. 1. Quadrupole
Mass Analyzer
A Quadrupole mass filter
consists of four parallel metal
rods with different charges
Two opposite rods have an
applied +ve potential and the
other two rods have a –ve
potential
The applied voltages affect
the trajectory of ions traveling
down the flight path
For given DC and AC
voltages, only ions of a certain
mass-to-charge ratio pass
through the quadrupole filter
and all other ions are thrown
out of their original path
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22. 2. TOF (Time of Flight)
Mass Analyzer
 TOF Analyzers separate
ions by time without the
use of an electric or
magnetic field.
In a crude sense, TOF is
similar to chromatography,
except there is no
stationary/ mobile phase,
instead the separation is
based on the kinetic energy
and velocity of the ions
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23. 3. Ion Trap Mass
Analyzer
It uses an electric
field for the separation
of the ions by mass to
charge ratios.
The electric field in
the cavity due to the
electrodes causes the
ions of certain m/z
values to orbit in the
space.
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24. 4. Fourier transform ion
cyclotron resonance (FT-
ICR)
 Uses a magnetic field
in order to trap ions into
an orbit inside of it.
 In this analyzer there
is no separation that
occurs rather all the ions
of a particular range are
trapped inside, and an
applied external electric
field helps to generate a
signal.
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25. WORKING PROCEDURE VIDEO OF
LC/MS
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26. Applications of LC-MS
Pharmaceutical Applications:
•Rapid chromatography of benzodiazepines
•Identification of bile acid metabolite
Biochemical Applications:
•Rapid protein identification using capillary LC/MS/MS and
database searching.
Clinical Applications:
•High-sensitivity detection of trimipramine and thioridazine 26
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27. Food Applications:
•Identification of aflatoxins in food
•Determination of vitamin D3 in poultry feed supplements
Environmental Applications:
•Detection of phenylurea herbicides
•Detection of low levels of carbaryl in food
Forensic Applications:
•illegal substances, toxic agents
•Explosives
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