advance technolgy of separation and dection sample in molecular range using combination of liquid chromatrography and mass spectrometry.

2. Chromatography is the collective term for a set of
laboratory techniques for the separation of mixtures. The
mixture is dissolved in a fluid called the mobile phase,
which carries it through a structure holding another
material called the stationary phase. The various
constituents of the mixture travel at different speeds,
causing them to separate. The separation is based on
differential partitioning between the mobile and
stationary phases. Subtle differences in a compound's
partition coefficient result in differential retention on the
stationary phase and thus changing the separation.
Chromatography was first developed and defined by the
Russion Botonist Mikhail Tswett in 1903. He produced a
colourful seperation of plant pigments using a column of
calcium carbonate(chalk).

3.  Modern techniques of mass spectrometry were
devised by Arthur Jeffrey Dempster and F.W. Aston
in 1918 and 1919 respectively.
 Mass spectrometry (MS) is an analytical chemistry
technique that measures the mass-to-charge ratio
(m/z) and abundance of gas-phase ions. A mass
spectrum (plural spectra) is a plot of the ion signal as
a function of the mass-to-charge ratio. The spectra are
used to determine the elemental or isotopic signature
of a sample, the masses of particles and of molecules,
and to elucidate the chemical structures of molecules,
such as peptides and other chemical compounds.

4.  Combining the two processes reduces the possibility of error, as it is
extremely unlikely that two different molecules will behave in the
same way in both a liquid chromatograph and a mass spectrometer.
 Therefore, when an identifying mass spectrum appears at a
characteristic retention time in a LC-MS analysis, it typically lends
to increased certainty that the analyte of interest is in the sample.

5. Schematic diagram.

6. MOBILE PHASE
COLUMN
ION SOURCE MASS ANALYSER ION CLLECTION
SYSTEM
VACCUM
SYSTEM
DATA HANDLING
SYSTEM
EFFUENT
Simple flow chart of LC-MS

7. LC-MS schematic
1
2

8. Instrumentation diagram..

9. To reduce separation time to achieve fast analysis.(From
hours to minutes)
HOW?
The solutes must move faster through stationary phase MEANS By
increasing the mobility of the liquid mobile phase.
We can achieve faster migration velocities of liquid mobile phase by-
1.Applying vacuum at the other end of the chromatographic column.
2.Applying high pressure on the liquid mobile phase.

10. Mordern LCMS Pumps..

11. • Column type.
• Specialized mode.
• The use of di-functional or tri-functional silanes
to create bonded groups with two or three
attachement points leading to phases with higher
stability in low or higher pH and lower bleed for
LCMS
• Most widely used columns for LCMS are:-
(1) fast LC column.
the use of short column. (15-50mm)
(2) Micro LC column.
the use of large column. ( 20-150mm)

12. Different Types of LCMS Columns..

13. • It is difficult to interface a liquid
chromatography to a mass-spectrometer
cause of the necessity to remove the solvent.
• The commnly used interface are:-
(1) Electrospray ionization (ESI)
(2) Thermospray ionization (TSI)
(3) Atmospheric pressure chemical ionization
(APCI)
(4) Atmospheric pressure photoionization
(APPI)
(5) Partical beam ionization.

16. 16
As soon as the solutes are eluted they should be detected and
quantitated with sensitive and specific or universal detectors.
With specific and universal detectors we have achieved the
following attributes:
High sensitivity.
High reliability and accuracy

17. QN504 Triple Quadrupole detector.

18. Varrious dectors used in LCMS..

19. Detectors And Lamps..

20.  They deflects ions 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) Ion trap
 (4) Fourier transform ion cyclotron
resonance (FT-ICR or FT-MS)

25. Modern LCMS (LCMS 2020 SHIMADZU)

26. Some parts of LCMS…

27. Updated LC Instrument..

28.  Molecular weight determination
 Determining the molecular weight of green
fluorescent proteins
 Structural determination e.g. structural
determination of ginsenoside.
 Pharmaceutical application e.g. identification
of bile acids metabolites.
 Biochemical application e.g. rapid protein
identification using capillary lc/ms/ms.

29. Food application e.g. identification of aflatoxin
in food determination of vitamin D3 in poultry
feed supplement using MS3
Environmental application e.g. detection of
phenyl urea herbicides, detection of low level
of carbaryl in food.

30. Thank U!