Chromatography report new

CHROMATOGRAPHY

 A laboratory technique in which the
components of a sample are separated
based on how they distribute between two
chemical or physical phases, one of which
is stationary and other of which is allowed
to travel through the separation system.

CHROMATOGRAPHY

 Introduced first by the Russian botanist
Mikhail Semenovich Tswett.
 Mixtures of solutes dissolved in a common
solvent are separated from one another by
a differential distribution of the solutes
between two phases.

PRINCIPLE

 Fractionalism of mixtures of
substances
 In the operation of the
chromatogram, a mobile gaseous or
liquid phase is use to wash the
substances to be separated through a
column of a porous material.

PRINCIPLE

 The rate of migration of the solute
depends upon the rate of interaction
of the solute with the two phases, one
being the mobile phases and the
other stationary phase as the
compounds travel through the
supporting medium.

DEFINITION OF TERMS:

 Capillary Action – the movement of liquid
within the spaces of a material due to the
forces of adhesion, cohesion, and surface
tension.
 Retention time-the average time of the
substance that is bound by the stationary
phase and will travel slowly through the
column and exit at some later time.
 Peak
 Viscosity

DEFINITION OF TERMS:

 VOCs – volatile organic compounds
 Stationary phase- fixed phase that is
coated or bonded within the column
 Mobile phase-phase that is flowing
through the column and causes
sample components to move towards
the column’s end.

COMPONENTS OF A CHROMATOGRAPH

 MOBILE PHASE – A phase that is flowing
through the column and causes sample
components to move toward the column’s end.
 STATIONARY PHASE- A fixed phase that is
coated or bonded within the column; it always
remain in the system.
It is responsible for delaying the movement of
compounds as they travel through the column.
 SUPPORT- onto which the SP is coated or
attached.

COMPONENTS OF A CHROMATOGRAPH
ORIGINAL SAMPLE
AND MOBILE PHASE
COLUMN

SUPPORT AND
STATIONARY
PHASE

Receiving
vessel

TYPES OF CHROMATOGRAPHY

 CAN BE CLASSIFIED
ACCORDING TO:
∞MOBILE PHASE
∞STATIONARY PHASE
∞SUPPORT

GAS CHROMATOGRAPHY

 A type of Chromatography in which the
mobile phase is a GAS.
 First GC system was developed by Erika
Cremer
 The presence of a gas mobile phase makes
GC valuable for separating substances like
VOCs that occur naturally as gases that can
easily be placed into gaseous phase.

GAS CHROMATOGRAPHY

• It can separate nanograms or
pictograms of volatile substances.

• It is principally a method for the
separation and quantitative
determination of gases and volatile
liquids and substances.

GAS CHROMATOGRAPHY

HOW IS IT PERFORMED?
 A System called Gas Chromatograph is used
to perform GC.
 COMPONENTS:
GAS SOURCE
INJECTION SYSTEM
COLUMN
DETECTOR

COLUMN

DETECTOR
MOBILE PHASE FLOW METER
SOURCE AND
FLOW CONTROL

INJECTOR

COLUMN OVEN

DATA
ACQUISITION

GAS CHROMATOGRAPHY
∞ GAS SOURCE- supplies the mobile phase. It is
typically a gas cylinder equipped with
pressure regulators to deliver the mobile
phase at a controlled state.

∞ INJECTION SYSTEM- consists of a heated
loop or port into which the sample is placed
and converted into a gaseous form.

GAS CHROMATOGRAPHY
∞ COLUMN- contains the stationary phase and
support material for the separation of
components in the sample. This column is
held in an enclosed area known as the column
oven.
∞ COLUMN OVEN- maintains the temperature
at a well-defined value.
∞ DETECTOR- monitors sample components as
they leave the column.

GAS CHROMATOGRAPHY
FACTORS THAT AFFECT GC:

 Requirements for the Analyte
 Volatility and Thermal Stability
 Chemical Derivatization

GAS CHROMATOGRAPHY
Common Mobile Phases in GC:
 Hydrogen
 Helium
 Nitrogen
 Argon

GAS CHROMATOGRAPHY
GC SUPPORT MATERIALS
 Packed Column
filled with small support particles that acts as an
adsorbent or that are coated with the desired stationary
phase.

 Open- Tubular Columns
stationary phase coated on or attached to its interior
surface.

GAS CHROMATOGRAPHY
GC STATIONARY PHASES

 Gas- Solid Chromatography ( solid adsorbents)
Gas-Liquid Chromatography (liquids coated on
solids)
Bonded phases

GAS CHROMATOGRAPHY
◊ Gas-Solid Chromatography
o Solid adsorbent is used as a stationary phase.
o Uses the same material as both the support and
stationary phase, with retention occurring through the
adsorption of analytes to the support’s surface.
o Example of support is a MOLECULAR SIEVE.
o Other supports include:
o ORGANIC POLYMERS - porous polystyrene
o INORGANIC SUBSTANCES – Silica or Alumina

GAS CHROMATOGRAPHY

o Increasing the surface area of the GSC support will
increase the phase ratio and result in higher retention for
analytes
o Pore size is important because only compounds smaller
than the pores are able to contact the surface are within
the space.
o Polarity of Support and its functional groups will also
affect how analytes will bind to them.

GAS CHROMATOGRAPHY
◊ Gas-Liquid Chromatography

o A chemical coating or layer is placed onto the support
and used as the stationary phase.

o Most Common types of GC.

o 100% dimethylpolysiloxane, 5%phenyl-95% methyl
polysiloxane – Examples of liquids that are used as
Stationary phase.

GAS CHROMATOGRAPHY
◊ Gas-Liquid Chromatography

o All of these liquids have High boiling points and low
volatilities, which allows them to stay within the
column at relatively high temperatures that are often
used in GC for sample injection and elution.

o Liquids are also wettable- easy to place onto a support
in a thin, uniform layer.

GAS CHROMATOGRAPHY
◊ Bonded Phases
o Column Bleed- most nonvolatile liquid will slowly
vaporize or break apart and leave the column over
time.
o Column bleed changes the retention characteristics of
the column.
o It can also cause some GC detectors to have a high
background and noisy signal as the stationary phase
leaves the column and enters the detector.
o Use of bonded phase minimize column bleed.

GAS CHROMATOGRAPHY
◊ Bonded Phases
o Produced by reacting groups on a polysiloxane
stationary phase with silanol groups that are
located on the surface of a silica support.

GAS CHROMATOGRAPHY
Types of Gas Chromatography Detectors
 General Detectors
x Thermal Conductivity Detector
-used for both organic and inorganic compounds
-measures the ability of the eluting carrier gas and
analyte mixture to conduct heat away from hot-wire
filament-”thermal conductivity”.
-example: Wheatstone bridge

GAS CHROMATOGRAPHY
 Flame Ionization Detector
- detects organic compounds by measuring
their ability to produce ions when they are burned
in flame.

GAS CHROMATOGRAPHY
 Selective Detectors
x Nitrogen-Phosphorous Detector
- selective for the determination of nitrogen- or
phosphorous containing compounds.
- Similar to FID, but does not use a flame for ion
production.

GAS CHROMATOGRAPHY
 Electron capture detector
- detects compounds that have electronegative
atoms or groups in their structure, such as halogen atoms
( I,Br,Cl and F) and Nitro Groups.
-can also be used to detect polynuclear aromatic
compounds, anhydrides and conjugated carbonyl
compounds.

GAS CHROMATOGRAPHY
Applications:
 Most effectively used for analyses of organic
compounds, space related, complex mixtures of
volatile substances at column temperature of
less than -40 °C to greater than 550° C.
 Geochemical research projects such as
determination of various environmental
pollutants at extremely low concentrations.

GAS CHROMATOGRAPHY

ADVANTAGES: DISADVANTAGES:
o Ability to provide qualitative o LIMITED to volatile
information and quantitative samples
information o Not suitable for
o FAST ANALYSIS thermally labile samples
o Efficient, providing high o Fairly difficult for large
resolution preparative samples
o Sensitive o Requires spectroscopy
usually mass
o Nondestructive
spectroscopy for
o Requires small samples confirmation of peak
o Inexpensive identity

LIQUID CHROMATOGRAPHY
√ A Chromatographic technique in which the
mobile phase is a liquid.
√ Originally developed by Russian botanist Mikhail
Tswett in 1903.
√ Its popularity is due to the ability of this method
to work directly with liquid samples, which
makes it valuable in such areas as food testing,
environmental testing and biotechnology.

HOW IS LIQUID CHROMATOGRAPHY
PERFORMED?
 A System known as a Liquid Chromatograph is
used to perform LC.

√ Components of the LC System:
 Support – enclosed in a Column
 Stationary phase- enclosed in a Column
 Liquid mobile phase-delivered to Column by means of
Pump
 Injection device- on analytical applications it is being
used,to apply samples to the column.
 Collection Device (optional)- placed after the column
to capture analytes as they elute.

FACTORS THAT AFFECT LIQUID CHROMATOGRAPHY:

*requires both a difference in retention and good efficiency
for it to separate two given chemicals
*Sample
*Analyte Requirements
*Formats
*Role played by the Mobile phase

Requirements for the Analyte:
Must be possible to place this chemical
into a liquid that can be injected onto the
column.
There must be a difference in retention
between the analytes to be prepared.

Types of Liquid Chromatography:
 Adsorption Chromatography
 Partition Chromatography
 Ion-Exchange Chromatography
 Size-Exclusion Chromatography
 Affinity Chromatography

1. ADSORPTION CHROMATOGRAPHY
A chromatographic technique that separates solutes
based on their adsorption to the surface of a support.
 Also known as Liquid-Solid Chromatography
 Equivalent method in GC is Gas-Solid Chromatography
 Uses the same material as both stationary phase and
support.
 Retention process can be explained on the ff below:
 A+ n M-Surface A-Surface + n M

 Elutropic strength- strength of a mobile
phase in adsorption chromatography
 It is a measure of how strongly a particular
solvent or liquid mixture will absorb to the
surface of a given support.
 Examples: silica and Alumina supports
 A liquid with large elutropic strength will
strongly adsorb to the given support, which
will prevent the analyte from binding to the
support.

 Stationary Phases and Mobile Phases
Silica (SiO2) - most popular support since it is
polar in nature, thus it will strongly retain polar
compounds.
Alumina (Al2O3) – general-purpose support, but
can retain some polar solutes so strongly that
they are irreversibly absorbed onto its surface.
Carbon-based Compounds- used as nonpolar
supports that retain nonpolar solutes and have a
strong mobile phase that is nonpolar.

 Applications
Help purify new compounds
Separation of geometrical isomers and
chemicals that belong to a given class of
substances
Remove undesired side-products during
synthesizing AZT or other drugs

2. PARTITION CHROMATOGRAPHY
o It is a Liquid Chromatographic technique in
which solutes are separated based on their
partitioning between a liquid mobile phase and a
stationary phase coated on a solid support.
o Support used is usually Silica
o Originally, it involves coating of support with a
liquid stationary phase that was immiscible with
the mobile phase

Two Main Categories of Partition Chromatography:
• Normal-phase- uses polar stationary phase

• Reversed phase-uses nonpolar stationary phase

Stationary Phases and Mobile Phases
 Liquid Stationary Phases coated onto solid supports
 Bonded Phases – widely used due to their better stability
and efficiency compared to liquid stationary phases.
 Silica - often used as the support. To place bonded phases
on this support, silanol groups on the surface of silica are
first treated with an organosilane that contains the
desired stationary phase as a side chain.
 Agents like triethylamine and trifluoroacetic acid can also
be added to the mobile phase to prevent silanol groups
from binding to analytes.

Applications:
Used in analytical laboratories
Separation of analytes in organic solvents and
chemicals that contain polar functional groups.
Employed in pharmaceutical industry as a
method for separating and analyzing drugs during
their testing and development.

3. ION- EXCHANGE CHROMATOGRAPHY
Solutes are separated by their adsorption
onto a support containing fixed charges on its
surface.
 Routinely used in Industry for the removal or
replacement of Ions in products.
 Used for the separation of charged compounds
( inorg. Ions, org. ions, AA, Proteins and Nucleic
Acids)

 Cation-exchanger – has negatively charged group
and is used to separate positive ions
 Anion-exchanger – has positively charged group
and is used to separate negative ions

Silica (SiO2) - most popular support since it is
polar in nature, thus it will strongly retain polar
compounds.
Polystyrene – used for small inorganic and organic
ions
Carbohydrate-based Gels – useful in separation of
biological compounds, which can have very
strong, undesirable binding to organic polymeric
resins like polysterene. Example: Agarose

 Applications
Removal of certain types of ions from samples such as
in water-purification
Direct separation and analysis of samples

4. SIZE-EXCLUSION CHROMATOGRAPHY
It is a LC technique that separates substances
based on different abilities of analytes to access
the mobile phase within the pores of a support..
o No true stationary phase is present in this system
o Uses a support that has a certain range of pore sizes.
o A separation based on size or molar mass.

Ideal support consists of a porous material
that does not interact directly with the injected
solute.
o Carbohydrate-based supports like dextran and and
agarose in their underivatized form can be used in SEC
for biological compounds and aqueous-based
samples.
o Polyacrylamide gel can also be used
o For organic solvents, Polystyrene can also be used
o Silica containing diol-bonded phase can be used on
aqueous samples

APPLICATIONS:
 Used in biological samples
 Transfer of large analytes from one solution to
another
 Removal of salts from sample
 Separation of biomolecules and polymers
 Estimation of molar mass

5. AFFINITY CHROMATOGRAPHY
A Technique based on biologically related
interactions.
o Makes use of the selective, reversible interactions that
characterize most biological systems.
o Example: binding of enzyme with its substrate.

Stationary phase is represented by the
affinity ligand.
o High specificity ligands- bind to only one or a few very
closely related molecules
o Examples:antibodies for binding to foreign agents and
single-stranded Nucleic Acids for separating and binding to
complemetary strands
o General ligands- bind to a family or class of related
molecules


Carbohydrate gels like agarose or
cellulose are commonly used with
affinity ligands serve as supports.

Silica can also be used with affinity
ligands

APPLICATIONS:
oLarge-Scale Purification method for
enzymes and proteins
oSample preparation
oDirect analysis of complex biological
samples.
oStudy of biological interactions.

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 Uses a pressure for the pumping of
aqueous or organic solution through a
column.
 The mobile phase is forced under
pressure through a long, narrow column,
yielding an excellent separation in a
relatively short time.
 Highly sensitive and specific.

CHROMATOGRAPHY
 Become the primary means of
monitoring the use of drugs and of
detecting drug abuse.
 Also used to separate the compounds
contributing to the fragrance of the
flowers.

CHROMATOGRAPHY
ADVANTAGES:
An automated process that takes only a few minutes
to produce results.
Uses gravity instead of high speed pump to force
compounds through the densely packed tubing.
Results are of high resolution and are easy to read.
Can be reproduce easily via automated process.

CHROMATOGRAPHY
DISADVANTAGES:
Difficult to detect coelution, which may lead to
inaccurate compound categorization.
High cost for equipment needed to conduct HPLC.
Operation is complex, requiring a trained technician
to operate.
Equipment has low sensitivity to some compounds
because of the speed of the process.

CHROMATOGRAPHY
Application:
 Use in biomedical research, routine
clinical determination and drug
researching programs

CHROMATOGRAPHY
 Instruments
 Fraction Collector
 Auto Sampler
 Pumping systems
 Columns & Packing
 Detectors
 Control Data & Processing

CHROMATOGRAPHY

CHROMATOGRAPHY
Application:
 Use in monitoring the use of therapeutic
drugs and detecting drug abuse.
 also use to separate compounds
contributing to the fragrance of flowers

OLD TECHNIQUES IN
CHROMATOGRAPHY
A separation that takes place on a flat
surface or a plane:

 PAPER CHROMATOGRAPHY

 THIN LAYER CHROMATOGRAPHY

PAPER CHROMATOGRAPHY
 Based on nature of solvent,
solubility of solute
and rate of diffusion.

 Uses paper as the
stationary phase and
a solvent as the
mobile phase.

 Solvent moves
through the paper by
a capillary action
 Separation depends
on the solubility of
solute and solvents,
the polarity of solvent,
and polarity of solutes
in the sample.

Visualization of the
separated sample
occurs by chemical
reaction, which
produces a color
change.

 Considered to be the simplest and
the most widely used of the
chromatographic techniques
because its APPLICABILITY TO THE
FOLLOWING:
 ISOLATION
 IDENTIFICATION
 AND QUANTITATIVE
DETERMINATION OF ORGANIC
AND INORGANIC COMPOUNDS

THIN LAYER CHROMATOGRAPHY
 Used as a semi-quantitative
screening test screening test
 Uses as thin layer of silica gel,
alumina gel, polyacrylamide gel, or
starch gel attached to glass plate as
stationary phase and the mobile
phase is liquid solvent.

 Fractions in the sample are generally
quite soluble in the solvent and move
with it up the stationary phase by
capillary action.
 Separated fractions are also developed
in TLC by applying a chemical reaction
with the separated fractions to produce
color changes


ADVANTAGES: DISADVANTAGES:
 Simple and economical  Spots are often faint
 Easy to perform since it
 TLC is difficult to
only involves spotting
the stationary phase reproduce
with the sample &  Not typically
placing one edge of the automated
stationary phase plate in
the mobile phase
reservoir.

Chromatography report new

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