2. CONTENTS :
INTRODUCTION
BASIC PRINCIPLE OF LC/MS
INSTRUMENTATION
COMPONENTS OF MASS SPECTROMETER
IONIZATION METHODS
MASS ANALYZER
DETECTORS
TANDEM MASS SPECTROMETRY
APPLICATIONS OF LCMS
FUTURE PROSPECTS OF LCMS
3. LIQUID CHROMATOGRAPHY
MASS SPECTROMETRY
INTRODUCTION:
Liquid chromatography-mass spectrometry (LC/MS)
is a technique that uses liquid chromatography (or
HPLC) with the mass spectrometry.
It is an analytical chemistry technique that
combines the physical separation capabilities of
liquid chromatography with the mass analysis
capabilities of mass spectrometry.
4. Basic Principle of LC/MS:
LC/MS combines the separating power of High
Performance Liquid Chromatography (HPLC), with
the detection power of Mass Spectrometry.
1. High Performance Liquid Chromatography
(HPLC):
Liquid chromatography involves two main
phases:
Mobile phase: Liquid (solvents i.e. ethanol,
Acetonitrile).
Stationary phase: Column packed with very
small particles
2. Mass Spectrometry :
Analytical technique that measures the mass-
to-charge ratio of charged particles.
5. Working Principle:
MS works by ionizing chemical compounds to
generate charged molecules or molecule fragments
and measuring their mass-to-charge ratios.
The components of the sample are ionized by one of
a variety of methods.
The ions are separated according to their mass-to-
charge ratio in an analyzer by electromagnetic field.
The ions are detected, usually by a quantitative
method. The ion signal is processed into mass
spectra.
9. Components of Mass
Spectrometer :
Ion source, which can convert gas phase sample
molecules into ions.
Analyzer, where ions are separated according to
their mass-to-charge ratio by applying
electromagnetic fields.
Detector, where ions are counted thus calculating
the abundances of each ion present.
10. Ionization Methods :
Following are the most common ionization methods :
i. Electrospray Ionization
ii. Atmospheric Pressure Chemical Ionization
iii. Atmospheric Pressure Photo-ionisation
Electrospray Ionization :
Method of molecule ionization used in the analysis of
multicomponent mixtures in an LC-MS system. It
belongs to a group of methods whereby “soft”
ionization is carried out under atmospheric pressure.
Electrospray ionization is a highly efficient technique
that does not cause the dissociation of the molecules
under study
11. . Under this method, the eluate stream leaving the
chromatographic column is introduced into the
ionization source through the capillary.
At the outlet of the capillary, the sample dissolved
in the solvent is exposed to a strong nebulizing
gas (typically, nitrogen) and a very strong electric
field, which results in the atomization of the
sample into charged microdroplets.
13. Atmospheric Pressure
Chemical Ionization:
Atmospheric pressure chemical ionization (APCI) is
a very similar technique to ESI. The manner of
sample ionization is the fundamental difference
between the two methods.
In APCI, the eluate leaving the chromatographic
column is heated and sprayed from the capillary
and then captured, in gaseous form, by a stream
of gas and carried to the electrode ions are
formed.
15. Atmospheric Pressure Photo-
ionization :
Atmospheric pressure photo-ionisation (APPI) uses
photons to excite and ionise molecules after
nebulisation.
The energy of the photons is chosen to minimise
concurrent ionisation of solvents and ion source gases.
The technique also gives predominantly singly-charged
ions and has been used for the analysis of neutral
compounds such as steroids and has been reviewed.
16. Mass Analyzer :
Its task is to separate ions in terms of their mass-to-
charge ratio and to direct the beam of focused ions to
the detector.
The key performance parameters of an analyzer
include;
(a) separation efficiency
(b) m/z measurement precision
(c) range of the m/z values measured
17. There are following kinds of mass analyzers that
can be used in LC/MS :
1. Quadrupole Analyzer:
The quadrupole analyzer (Q) consists of four
metal electrodes in the form of symmetrically
arranged rods. It works like a mass filter that, with
the specific parameters of the electromagnetic
field, allows the passage of ions with the selected
mass-to-charge ratio values, while other ions are
dispersed and do not reach the detector.
18. Time-of-Flight Analyzer
The time-of-flight mass analyzer (TOF) consists of
an ion accelerating grid and a flight tube (about 1 m
long), through which the ions travel to the detector.
The analyzer separates ions accelerated by an
electric field according to their velocity which
depends on their mass and charge.
19. Ion Trap Analyzer:
One of the most popular ion trap analyzers (IT) is
the quadrupole ion trap consisting of a ring-shaped
electrode and two electrodes with a spherical
cross-section, with the space between them
forming a trap. The ion trap analyzer traps ions
with a specific mass-to-charge ratio by means of
an electric field.
20. Detectors :
The detector is used to count the ions emergent from
the mass analyzer, and may also amplify the signal
generated from each ion. Following are three different
kinds of detectors are used in Mass Spectrometry;
Electron Multipliers: used to convert either –ve, +ve
ions into electrons, that will be amplified and detected.
21. Dynolyte Photomultiplier:
The dynode of Dynolyte photomultipliers converts
the charged ions into electrons. These electrons
stick to a phosphor and emit photons, and that
photons are made to strike the photomultiplier to
achieve multiplied signals for recording.
22. Micro Channel Plates: Microchannel Plate
(MCP) is commonly employed in ToF
spectrometers. This will have very low time
response and high degree of sensitivity.
23. Interface :
The interface is most often an electrospray ion
source; however atmospheric pressure chemical
ionization interface is also used. Various techniques
of deposition and drying have also been used such
as using moving belts; however the most common of
these is off-line MALDI deposition.
LC/ MS for Detailed Structure-Dependent
Analysis:
It is hard in HPLC to be certain about purity of a
particular peak, and if it contains only a single chemical.
Adding a Mass Spectrometry to this will tell you the
masses of all the chemicals present in the peak, which
can be used for identifying them, and an excellent method
to check for the purity.
24. Tandem Mass Spectrometry
Tandem mass spectrometry (MS/MS) is a system of two
combined analyzers of the same type or different types,
characterized by high separation efficiency.
The ions produced by the source are separated in the
first analyzer (MS1). Ions with the selected m/z value
reach the collision cell where, depending on the analysis
conditions, they undergo dissociation or remain
unchanged.
In comparison with analysis using a single analyzer,
tandem analysis shows a considerable improvement in
selectivity and considerably increased sensitivity.
25. APPLICATIONS OF LCMS :
Molecular Weight Determination
Structural Elucidation
Pharmaceutical Applications
Food and Environmental Applications
Characterization and Identification of Compounds
Carotenoids
Proteomics
Glycopeptides Characterization
Peptide Mapping
• Bioanalysis of various Biological Samples
26. Analysis of Complex Lipid Mixtures :
It is a LC-MS-based methodology for the investigation of
lipid mixtures where it has described, and its application to
the analysis of human lipoprotein-associated lipids is
demonstrated.
Phytoconstituents:
LC–MS provides a tool for differentiating this immense
plant biodiversity due to this technique’s capability of
analyzing a broad range of metabolites and highly polar
and/or higher molecular weight molecules (oligosaccharides
and lipids).
27. FUTURE PROSPECTS OF
LCMS
• Metabolomics :
The next 5–10 years will inevitably witness increased
inter-laboratory cooperation in order to collate as much LC-
MS-based metabolite data as possible. The integration of
NMR to LC-MS-based metabolic profiling and metabolomic
studies will likely increase, either through the offline
analysis of collected LC fractions or through hybrid LC-
NMR-MS instrumentation.
• Proteomics:
performance of proteomic experiments.
Cleanness of samples in relation to non-protein
contaminants .
affects the protein identification rate.
28. Pharmacovigilance:
Referred to as Drug Safety
Relates to the collection, detection, assessment,
monitoring, and also prevention of adverse side
effects with pharmaceutical products.
The detection and monitoring can be done by LC-
MS based disease modifying technique which
provides detailed profiles.
29. Organic/Inorganic Hybrid
Nanoflowers
Helps in the development of drug delivery
systems, biosensors, biocatalysts, and bio - related
devices is anticipated to take multiple directions.
The application of nanoflowers in bio-catalysis and
the design of highly sensitive bio-sensing kits, as
well as industrial bio-related devices with advanced
functions should receive increasing attention.
30. References:
Kumar, P. R., & Rini, R. (2016). LCMS-A REVIEW
AND A RECENT UPDATE.
Stachniuk, A., & Fornal, E. (2016). Liquid
Chromatography-Mass Spectrometry in the Analysis of
Pesticide Residues in Food. Food Analytical
Methods, 9(6), 1654-1665.
Parasuraman, S., Anish, R., Balamurugan, S.,
Muralidharan, S., Kumar, K. J., & Vijayan, V. (2014).
An Overview of Liquid Chromatography-Mass
Spectroscopy Instrumentation. Pharmaceutical
Methods, 5(2), 47.
