A brief description about the underlying principles, its governing laws, methods & applications.

1. CHROMATOGRAPHY
AND
MASS
SPECTROSCOPY
ROHAN DEOKAR

2. WHAT IS CHROMATOGRAPHY???
 Quite simply, it is a broad range of physical methods used to
separate and or to analyze complex mixtures.
 The mixture is dissolved in a fluid called the mobile phase/eluent,
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.

3. HOW DOES IT WORK ?????
 A mixture of various components enters the chromatography
process, and the different components are flushed through the
system at different rates.
 These differential rates of migration as the mixture moves over
adsorptive materials provide separation.
 Repeated sorption/desorption acts that take place during the
movement of the sample over the stationary bed determine the
rates.
 The smaller the affinity a molecule has for the stationary phase,
the shorter the time spent in a column.

4. TERMS USED IN CHROMATOGRAPHY
 The analyte/sample/solute is the substance to be separated during
chromatography which may consist of a single component or a
mixture of components.
 Analytical chromatography is used to determine the existence and
possibly also the concentration of analyte(s) in a sample.
 A bonded phase/stationary phase is covalently bonded to the support
particles or to the inside wall of the column.
 The eluate is the mobile phase leaving the column.
 The retention time is the characteristic time it takes for a particular
analyte to pass through the system (from the column inlet to the
detector) under set conditions.

5. BASIC OPERATION
 The process of a chromatographic separation
takes place within a hollowed center
chromatography column made of glass/metal in
a packed bed or open tubular column.
 A packed bed column contains particles of the
stationary phase while open tubular columns
are lined with a thin film stationary phase.
 The mobile phase/carrier fluid is a solvent
which carries the mixture to be separated and
can either be a liquid or a gas, depending on
the type of process, while the stationary phase
is a viscous liquid coated on the surface of
solid particles.
 The partitioning of solutes between the two
phases lead to the desired separations.
 The materials comprising the mobile and
stationary phases vary depending on the type
of chromatographic process being performed.

6. WORKING MECHANISM
1. FEED INJECTION –
The feed is injected into the mobile phase. The mobile phase flows through
the system by the action of a pump.
2. SEPARATION IN THE COLUMN -
As the sample flows through the column, its components will adsorb to the
stationary phase to varying degrees. Those with strong attraction to the
support move slowly than those with weak attraction.
3. ELUTION FROM THE COLUMN-After
the sample is flushed or displaced from the stationary phase, the
different components will elute from the column at different times.
The components with the least affinity for the stationary phase (weakly
adsorbed) will elute first, while those with the greatest affinity for the
stationary phase (strongly adsorbed) will elute last.
4. DETECTION -
The different components are collected as they emerge from the column.
A detector analyzes the emerging stream by measuring a property which is
related to concentration and characteristic of chemical composition.

8. THE CHROMATOGRAM
 Since the sample is separated in the
column, different peaks correspond
to different components present.
 The chromatogram show the results
of separations of protein mixtures by
ion exchange chromatography.
 The lettered peaks correspond to
different proteins (A = ovalbumin, B =
conalbumin, C = cytochrome c, D =
lysozyme).
 The separation corresponding to the
chromatogram on the left was
performed at pH 5.85, while the one
on the right was performed at pH 6.5.
 Operation conditions such as pH and
temperature have a significant effect
on the output.

9. WHAT INFORMATION CAN BE ATTAINED ???
 The level of complexity of the sample is indicated by
the number of peaks which appear.
 Qualitative information about the sample composition is
obtained by comparing peak positions with those of
standards.
 Quantitative assessment of the relative concentrations
of components is obtained from peak area
comparisons.
 Column performance is indicated by comparison with
standards.

10. WHAT IS MASS SPECTROSCOPY?????
 Mass spectrometry (MS) is an analytical technique that separates the
components of a sample by their mass, producing spectra of the
masses of the molecules of a sample material.
 The spectra determine the elemental composition of a sample, the
masses of particles and molecules, and to explain the chemical
structures of molecules and protein characterization.
 It is mainly used in identifying unknown compounds, determining
isotopic composition of elements in a molecule, and determining
the structure of a compound by observing its fragmentation.
 Other uses include quantifying the amount of a compound or studying
the fundamentals of gas phase ion chemistry (the chemistry of ions
and neutrals in a vacuum).
 Biomedical applications include use for respiratory gas and trace
analysis.

11. BASIC PRINCIPLE
 The sample is vaporized into a gas and then ionized.
 Ions are separated according to their mass to charge ratio and are
accelerated through a potential difference and focused into a beam.
 The ion beam passes through a magnetic field which bends the
charged stream.
 Lighter components or components with more ionic charge will
deflect in the field more than heavier or less charged components.
 A detector counts the number of ions at different deflections and the
data can be plotted as a 'spectrum' of different masses.
 The atoms or molecules can be identified by correlating known
masses to the identified masses or through a characteristic
fragmentation pattern.
 Signal processing results are displayed as spectra of the relative
abundance of ions as a function of the mass-to-charge ratio.

12. COMPONENTS OF THE SPECTROMETER
A mass spectrometer consists of three components: an ion source, a
mass analyzer, and a detector.
1. ION SOURCE / IONIZER -
 The ion source is the part of the mass spectrometer that ionizes the
material under analysis (the analyte).
 There are a wide variety of ionization techniques, depending on the
phase (solid, liquid, gas) of the sample and the efficiency of various
ionization mechanisms for the unknown species and determine the
types of samples which can be analyzed.
 Electron ionization and chemical ionization are used
for gases and vapors while electrospray ionization and matrix-assisted
laser desorption/ionization (MALDI) are used
for liquid and solid biological samples.
 The ions are then transported by magnetic or electric fields to the
mass analyzer.

13. 2. MASS SELECTION -
 Mass analyzers separate the ions according to their mass-to-charge
ratio. The following two laws govern the dynamics of charged
particles in electric and magnetic fields in vacuum:
 (Lorentz force law);
 (Newton's second law of motion)
 F is the force applied to the ion, m is the mass of the ion, a is the
acceleration, Q is the ion charge, E is the electric field, and v × B is
the vector cross product of the ion velocity and the magnetic field.
 Equating the above expressions for the force applied to the ion yields
the classic equation of motion for charged particles:
3. DETECTOR-The
final element of the mass spectrometer is the detector which
records either the charge induced or the current produced when an
ion passes by or hits a surface. Some detectors also give spatial
information.

14. BASIC WORKING OF A MASS SPECTROMETER
 The following example of sodium
chloride describes the working
operation.
 In the ion source, the sample
is vaporized and ionized (transformed
into electrically charged particles)
into sodium (Na+) and chloride(Cl-) ions.
 Sodium atoms and ions are
monoisotopic, with a mass of about 23
amu. Chloride atoms and ions come in
two isotopes with masses of
approximately 35 and 37 amu.
 The analyzer part of the spectrometer
contains electric and magnetic fields,
which exert forces on ions traveling
through these fields.

15. S  The speed and direction of a
charged particle may be varied while
passing through the electric field and
magnetic field respectively.
 The magnitude of the deflection of
the moving ion's trajectory depends
on its mass-to-charge ratio. Lighter
ions get deflected by the magnetic
force more than heavier ions.
 The streams of sorted ions pass
from the analyzer to the detector,
which records the relative
abundance of each ion type.
 This information is used to
determine the chemical element
composition of the original sample
and the isotopic composition of its
constituents (the ratio of 35Cl to 37Cl).

16. ADVANTAGES AND DISADVANTAGES
Advantages include-
1. Increased sensitivity since the analyzer reduces background
interference.
2. Excellent specificity from characteristic fragmentation
patterns to identify unknowns or confirm the presence of
suspected compounds.
3. Information about molecular weight and isotopic abundance
of elements.
Disadvantages include-
1. It fails to distinguish between optical and geometrical
isomers and the positions of substituent in o-, m- and p-positions
in an aromatic ring.
2. Limited scope in identifying hydrocarbons that produce
similar fragmented ions.

17. COMBINED TECHNIQUES
 An important enhancement to the mass resolving and mass
determining capabilities of mass spectrometry is using it in
tandem with chromatographic separation techniques.
 A common combination is gas chromatography-mass
spectrometry (GC/MS or GC-MS) in which a gas
chromatograph is used to separate different compounds.
 This stream of separated compounds is fed online into the ion
source, a metallic filament to which voltage is applied.
 This filament emits electrons which ionize the compounds.
The ions can fragment, yielding predictable patterns.
 Intact ions and fragments pass into the mass spectrometer's
analyzer and are eventually detected.
 These technique are used in the fields of forensics and
determination of respiratory gas analysis along with trace
analysis.

18. THANK YOU !!!!!!