Simple separation VS Chromatographic
separation
Principle of simple separation (exp partitioning
between phases):
- The separation occurs only one direction
- Increase efficiency by using fresh extracting phase.
The principle of chromatographic separation:
extracting the solutes back and forth between fresh
portion of the two phases. The two phases used are
called the mobile phase and the stationary phase.
Chromatography


1)
2)
Chromatography is the separation of a
mixture based on the different degrees
to which they interact with two
separate material phases:
The two phases are:
The stationary phase
The mobile phase
The stationary phase – a phase that is fixed
in place either in a column or in a planar
surface. The stationary phase is either a
porous solid used alone or coated with a
stationary liquid phase.
 The mobile phase – a phase that moves
over or through the stationary phase, carrying
with it the analyte mixture.
It is also called the eluting fluid. The mobile
phase can be a gas, a liquid, or a supercritical
fluid.

Column VS Planar
chromatography

Principle of column
chromatography:
The stationary phase is placed in a
narrow column through which the mobile
phase moves under the influence of
gravity or pressure.

Principle of planar chromatography
The stationary phase coats a flat glass, metal,
or plastic plate and is placed in a developing
chamber. A reservoir containing the mobile
phase is placed in contact with the stationary
phase, and the mobile phase moves by
capillary action.
Exp paper chromatography and Thin layer
chromatography.
Column Chromatography and
Planar Chromatography
Separation column
Paper or a
substrate
coated with
particles
Packing material
Column Chromatography
Paper Chromatography
Thin Layer Chromatography (TLC)
Output
concentration
Separation Process and Chromatogram
for Column Chromatography
Chromatogram
Time
7
Intensity of detector signal
Chromatogram
tR
tR : Retention time
Peak
t0
t0 : Non-retention time
h
A
A : Peak area
h : Peak height
Time
8
1. Retention factor = the degree of the retention of the sample component in
column k is defined as the time the solute resides in the stationary phase (ts)
relative to the time it resides in the mobile phase (tm).
2. Number of plates (N) is the number of plates of a column toward a particular
compound.
3. Height of plate, (H) is the plate height or the length of a column (L) divided
by the number of theoretical plates
4. Separation factor of selectivity factor, a for solutes A and B is defined as the
ratio of the distribution constant of the more strongly retained solute (B) to the
distribution constant of the less strongly held solute (B).
5. Resolution of a chromatographic column is a quantitative measure of its
ability to separate analytes A and B
or
Chromatographic Techniques

1.
-
-
Classified according to the type of equilibration
process involved. Equilibration process control by the
type of stationary phase.
Adsorption Chromatography
The stationary phase is a solid.
Sample components are adsorbed on the solid
stationary phase.
The mobile phase may be a liquid or a gas.
The sample components are distribute between the
two phases through a combination of sorption and
desorption process.
Example: Thin layer chromatography.
Chromatographic Techniques
2. Partition Chromatography
- The stationary phase is a liquid supported on an
inert solid.
- The mobile phase may be a liquid or a gas.
- In normal-phase C: a polar SP and non-polar MP
is used for nonpolar solutes.
- In reversed-phase C:nonpolar SP and polar MP
is used for polar solutes.
- Reversed phase is the most widely used.
Chromatographic Techniques
3. Ion Exchange and 4. Size Exclusion Chromatography
Stationary Phase
Mechanism
Ion Exchange Ion exchange resin ion exchange
equilibria
Size
A sievelike structure penetration of
Exclusion
a certain size
Chromatogra
phy
GAS CHROMATOGRAPHY (GC)
A chromatographic technique in which
the mobile phase is a gas.
 Parts of GC are:
The mobile phase
The stationary phase
Sample introductions
Temperature control
Detectors

Principle of Gas Chromatography
Sample should be converted to vapor state (if it is not
already a gas).
 Separation occurs as the vapor constituents equilibrate
between carrier gas and the SP.
 The sample is automaticaly detected by detector.
 Measuring the retention time and comparing this time
with that of a standard of a pure substances make it
possible to identify the peak.
 Since the area of the peak is porpotional to the
concentration, and so the amount of the substance can
be quantitatively determined.
 The peak height can be compared with a calibration
curved prepared in a same manner.

Importance parts of GC
There are three parts most important for
GC
1. The columns
2. The detectors
3. The mobile phase gas supply
The Columns
Commonly used columns are packed
columns and capillary columns.
Packed Column
 About 1-10 m long and 0.2-0.6 cm in
diameter.
 Short columns made of glass and longer
columns made of stainless steel or can
also made of Teflon.

Functions of GC Columns
To contain the stationary phase and the
passing way of the mobile phase.
 The site where the separation of analyte
occurs.
 To provide analysis in terms of resolution,
sensitivity and retention time.

The Detectors
Over 40 detectors have been developed since
the introduction of GC.

Commonly used detectors
1. Thermal Conductivity Detector (TDC)
- The original detector
2. Flame Ionization Detector (FID)
- The most sensitive and widely used detector for
organic compounds.
3. Flame Photometric Detectors (FPD)

Functions of Detectors
To respond to compounds analysed.
 To automatically detect the sample as it
emerges from the column.

The mobile phase gas supply
Usually an inert gas that available in pure form such
as argon, helium or nitrogen.
 A highly dense gas is more effective.
 The choice of gas determine by the type of detector.
Functions:
- To bring along gas and injected compounds
throughout the column up to detector.
- To provide equilibration between the carrier gas and
the stationary phase for compounds separation.


-
1.
2.
3.
Sample Introduction
Must consider three rules:
All constituents injected into GC must be
volatile.
The analyte must be present at an
appropriate concentration.
Injecting the sample must not degrade the
separation (thermally stable).
Volatile Sample
 A volatile compound is a compound that easily
evaporated because of their low molecular
weight.
 In GC, the sample constituents need to be
volatiled in order to move through the
column.
 Nonvolatile solutes will condense on the
column, degrading the column’s performance.
Exp. of volatile compounds are from the
monoterpenoids group (limonene, linalool,
champor, menthol etc.)
Applications of GC
 Widely used for the analysis of diverse array
of samples in environmental, clinical,
pharmaceutical, biochemical, forensic, food
science and petrochemical laboratories.

GC – Mass Spectrometry (GC-MS)
GC-MS is a sophisticated instrumental technique that produces,
separates, and detects ion in the gas phase.
Today, relatively inexpensive compact benchtop system are available
and widely used in laboratories.
GC – Mass Spectrometry (GC-MS)
• Mass spectroscopy is used to determine
the molecular formula of the unknown
compound.
• Mass spectroscopy data that provides
structural information tends to be
unreliable and thus will only be used to
verify a possible structure or in the
event that the other spectral techniques
are unsuccessful.
Principle of GC-MS
Block diagram of mass spectroscopy
• The inlet transfers the sample into the vacuum of the mass
spectrometer. In the source region, neutral sample molecules are
ionized and then accelerated into the mass analyzer.
• The mass analyzer is the heart of the mass spectrometer. This
section separates ions, either in space or in time, according to their
mass to charge ratio.
• After the ions are separated, they are detected and the signal is
transferred to a data system for analysis.
• All mass spectrometers also have a vacuum system to maintain the
low pressure, which is also called high vacuum, required for operation.
• High vacuum minimizes ion-molecule reactions, scattering, and
neutralization of the ions.
• In some experiments, the pressure in the source region or a part of
the mass spectrometer is intentionally increased to study these ionmolecule reactions. Under normal operation, however, any collisions will
interfere with the analysis.
Ionization Source
Electron Impact (EI)
Electron Ionization (EI) is the most common
ionization
technique
used
for
mass
spectrometry. EI works well for many gas phase
molecules, but it does have some limitations.
Although the mass spectra are very
reproducible and are widely used for spectral
libraries, EI causes extensive fragmentation so
that the molecular ion is not observed for many
compounds. Fragmentation is useful because it
provides
structural
information
for
interpreting unknown spectra.
Electron Ionization Source
Electron Ionization Process
A) Ionizing electron approaches the electron cloud of a
molecule
B)Electron cloud distorted by ionizing electron
C) Electron cloud further distorted by ionizing electron
D) Ionizing electron passes by the molecule
E) Electron cloud of molecule ejecting an electron
F) Molecular ion and ejected electron.
Chemical Ionization
Chemical Ionization (CI) is a “soft” ionization
technique that produces ions with little excess
energy. As a result, less fragmentation is observed
in the mass spectrum.
Since this increases the abundance of the molecular
ion, the technique is complimentary to 70 eV EI.
CI is often used to verify the molecular mass of an
unknown. Only slight modifications of an EI source
region are required for CI experiments.
In Chemical Ionization the source is enclosed in a small cell with
openings for the electron beam, the reagent gas and the sample.
The reagent gas is added to this cell at approximately 10 Pa (0.1 torr)
pressure.
This is higher than the 10-3 Pa (10-5 torr) pressure typical for a mass
spectrometer source. At 10-3 Pa the mean free path between
collisions is approximately 2 meters and ion-molecule reactions are
unlikely.
In the CI source, however, the mean free path between collisions is
only 10-4 meters and analyte molecules undergo many collisions with
the reagent gas.
The reagent gas in the CI source is ionized with an electron beam to
produce a cloud of ions. The reagent gas ions in this cloud react and
produce adduct ions like CH5+ ,which are excellent proton donors.
When analyte molecules (M) are introduced to a source region with
this cloud of ions, the reagent gas ions donate a proton to the analyte
molecule and produce MH+ ions.
The energetic of the proton transfer is controlled by using different
reagent gases.
The most common reagent gases are methane, isobutane and ammonia.
Methane is the strongest proton donor commonly used with a proton
affinity (PA) of 5.7 eV. For softer ionization, isobutane (PA 8.5 eV)
and ammonia (PA 9.0 eV) are frequently used.
Acid base chemistry is frequently used to describe the chemical
ionization reactions. The reagent gas must be a strong enough
Brønsted acid to transfer a proton to the analyte.
Fragmentation is minimized in CI by reducing the amount of excess
energy produced by the reaction. Because the adduct ions have little
excess energy and are relatively stable, CI is very useful for
molecular mass determination.
Mass Analyzer
After ions are formed in the source region they
are accelerated into the mass analyzer by an
electric field. The mass analyzer separates these
ions according to their m/z value.
The selection of a mass analyzer depends upon the
resolution, mass range, scan rate and detection
limits required for an application.
Each analyzer has very different operating
characteristics and the selection of an instrument
involves important tradeoffs.
Quadrapole Mass Filter
The quadrupole mass spectrometer is the most
common mass analyzer.
Its compact size, fast scan rate, high transmission
efficiency, and modest vacuum requirements are
ideal for small inexpensive instruments.
It ‘filter’ and only allow specific ions to pass.
Most quadrupole instruments are limited to unit
m/z resolution and have a mass range of m/z 1000.
Many benchtop instruments have a mass range of
m/z 500 but research instruments are available
with mass range up to m/z 4000.
Quadrapole Mass Analyzer
•Only compound with specific m/z ratio will resonate along the field
(stable path)
•Achieved by rapidly varying the voltage
Time of Flight Analyzer
The time-of-flight (TOF) mass analyzer separates ions in time as
they travel down a flight tube.
This is a very simple mass spectrometer that uses fixed voltages
and does not require a magnetic field. The greatest drawback is
that TOF instruments have poor mass resolution, usually less than
500.
These instruments have high transmission efficiency, no upper m/z
limit, very low detection limits, and fast scan rates.
For some applications these advantages outweigh the low resolution.
Recent developments in pulsed ionization techniques and new
instrument designs with improved resolution have renewed interest
in TOF-MS.
This picture shows the working principle of a linear time of flight mass
spectrometer.
To allow the ions to fly through the flight path without hitting anything
else, all the air molecules have been pumped out to create an ultra high
vacuum.
Ions of different m/z ratio travel at different velocities.
The difference in arrival times separate two (or more) ions.
Graph of ion intensity versus mass-to-charge ratio (m/z)
(units daltons, Da)
Liquid Chromatography
Chromatography in which the mobile
phase is a liquid.
◦ The liquid used as the mobile phase is
called the “eluent”.
 The stationary phase is usually a solid or
a liquid.
 In general, it is possible to analyze any
substance that can be stably dissolved in
the mobile phase.

43
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Analyze sample in liquid form.
The sample carried through a chromatographic
column by a liquid mobile phase.
 Parts of HPLC are:
HPLC column
The mobile phases
The stationary phases
Sample introductions
HPLC plumbing
Detectors


High Performance Liquid
Chromatography
http://www.waters.com/WatersDivision/ContentD.asp?watersit=JDRS-6UXGYA&WT.svl=1
From Liquid Chromatography to High
Performance Liquid Chromatography


Higher degree of separation!
 Refinement of packing material (3 to 10 µm)
Reduction of analysis time!
 Delivery of eluent by pump
 Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid
chromatography!
47
Flow Channel Diagram for High
Performance Liquid Chromatograph
Detector
Column
Pump
Eluent
(mobile phase)
Sample injection unit
(injector)
Degasser
Column oven
(thermostatic column
chamber)
Drain
Data processor
http://www.lcresources.com/resources/getstart/2c01.htm
What’s the different between
HPLC and GC?
Solvent Delivery Pump
A solvent delivery pump that can maintain a constant, non-pulsating
flow of solvent at a high pressure against the resistance of the
column is required.
Sample Injection Unit
There is a high level of pressure between the pump and the column;
a device that can inject specific amounts of sample under such
conditions is required.
Column
The technology for filling the column evenly with refined packing
material is required. Also, a material that can withstand high
pressures, such as stainless steel, is required for the housing.
Detector
Higher degrees of separation have increased the need for highsensitivity detection, and levels of sensitivity and stability that can
respond to this need are required in the detector.
Detection in HPLC
*There are six major HPLC detectors:






Refractive Index (RI) Detector
Evaporative Light Scattering Detector (ELSD)
UV/VIS Absorption Detectors
The Fluorescence Detector
Electrochemical Detectors (ECDs)
Conductivity Detector
* The type of detector
utilized depends on
the characteristics
of the analyte of
interest.
http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4
Refractive Index Detector


Based on the principle that every transparent
substance will slow the speed of light passing
through it.
◦ Results in the bending of light
as it passes to another material
of different density.
◦ Refractive index = how much
the light is bent
The presence of analyte molecules
in the mobile phase will generally
http://farside.ph.utexas.edu/teaching/3
change its RI by an amount almost 02l/lectures/img1154.png
linearly proportional to its
concentrations.
Refractive Index Detector






Affected by slight changes in mobile phase
composition and temperature.
Universal-based on a property of the mobile phase
It is used for analytes which give no response with
other more sensitive and selective detectors.
◦ RI = general
 responds to the presence of all solutes in the
mobile
phase.
Reference= mobile phase
Sample= column effluent
Detector measures the
differences between the RI of
the reference and the sample.
http://hplc.chem.shu.edu/HPLC/index.html
Evaporative Light Scattering
Detector (ELSD)


Analyte particles don’t scatter light
when dissolved in a liquid mobile phase.
Three steps:
1) Nebulize the mobile phase effluent
into droplets.
 Passes through a needle and mixes
with hydrogen gas.
2) Evaporate each of these droplets.
 Leaves behind a small particle of
nonvolatile analyte
3) Light scattering
 Sample particles pass through a cell
and scatter light from a laser beam
which is detected and generates a
signal.
http://www.sedere.com/WLD/whatis.html
HPLC Chromatogram
Types of HPLC
There are numerous types of HPLC which
vary in their separation chemistry.
◦ All chromatographic modes are possible:
 Ion-exchange
 Size exclusion
 Also can vary the stationary & mobile
phases:
◦ Normal phase HPLC
◦ Reverse phase HPLC

Chromatographic Modes of HPLC


Ion exchange:
◦ Used with ionic or ionizable samples.
◦ Stationary phase has a charged surface.
 opposite charge to the sample ions
◦ The mobile phase = aqueous buffer
◦ The stronger the charge on the analyte, the more it will be
attracted to the stationary phase, the slower it will elute.
Size exclusion:
◦ Sample separated based on size.
◦ Stationary phase has specific pore sizes.
◦ Larger molecules elute quickly.
◦ Smaller molecules penetrate inside the pores of the
stationary phase and elute later.
Normal Phase HPLC
Stationary phase: polar, silica particles
 Mobile phase: non-polar solvent or mixture
of solvents
 Polar compounds:
◦ Will have a higher affinity for the polar,
stationary phase
◦ Will elute slower
 Non-polar compounds:
◦ Will have a higher affinity for the nonpolar, mobile phase
◦ Will elute faster

Reverse Phase HPLC

Stationary phase: non-polar
◦ Non-polar organic groups are covalently attached to the silica
stationary particles.
 Most common attachment is a long-chain
n-C18 hydrocarbon
 Octadecyl silyl group, ODS

Mobile phase: polar liquid or
of liquids
Polar analytes will spend more time
polar mobile phase.
◦ Will elute quicker than non-polar analytes


Most common type of HPLC used today.
mixture
in the
http://www.lcresources.com/
resources/getstart/3a01.htm
Advantages of High Performance
Liquid Chromatography






High separation capacity, enabling the batch
analysis of multiple components
Superior quantitative capability and
reproducibility
Moderate analytical conditions
◦ Unlike GC, the sample does not need to be
vaporized.
Generally high sensitivity
Low sample consumption
Easy preparative separation and purification of
samples
59
Fields in Which High Performance
Liquid Chromatography Is Used


Biogenic substances
◦ Sugars, lipids, nucleic acids,
amino acids, proteins,
peptides, steroids, amines,
etc.
Medical products
◦ Drugs, antibiotics, etc.



Food products
◦ Vitamins, food
additives, sugars,
organic acids, amino
acids, etc.
Environmental samples
◦ Inorganic ions
◦ Hazardous organic
substances, etc.
Organic industrial
products
◦ Synthetic polymers,
additives, surfactants,
etc.
60
HPLC Applications




Can be used to isolated and purify compounds for
further use.
Can be used to identify the presence of specific
compounds in a sample.
Can be used to determine the concentration of a
specific compound in a sample.
Can be used to perform chemical separations
◦ Enantiomers (mirror image molecular structure)
◦ Biomolecules
HPLC Applications
*HPLC has an vast amount of current & future applications*

Some uses include:
◦ Forensics: analysis of explosives, drugs, fibers, etc.
◦ Proteomics: can be used to separate and purify protein
samples
 Can separate & purify other biomolecules such as:
carbohydrates, lipids, nucleic acids, pigments, proteins,
steroids
◦ Study of disease: can be used to measure the presence &
abundance of specific biomolecules correlating to disease
manifestation.
◦ Pharmaceutical Research: all areas including early
identification of clinically relevant molecules to large-scale
processing and purification.
Ion Exchange Chromatography
The principle
- To separate inorganic ions, both cations
and anions.
- Separate based on exchange of ions in the
stationary phase.
- The stationary phase consists of beads
made of polystyrene polymer crosslinked
with divinylbenzene.
Electrophoresis
Electrophoresis
• Electrophoresis is a separations technique that is based on
the mobility of ions in an electric field.
• Positively charged ions migrate towards a negative
electrode and negatively-charged ions migrate toward a
positive electrode.
• For safety reasons one electrode is usually at ground and
the other is biased positively or negatively.
• Ions have different migration rates depending on their
total charge, size, and shape, and can therefore be
separated.
Electrophoresis
• An electrode apparatus consists of a high-voltage supply, electrodes,
buffer, and a support for the buffer such as filter paper, cellulose
acetate strips, polyacrylamide gel, or a capillary tube.
• Open capillary tubes are used for many types of samples and the
other supports are usually used for biological samples such as protein
mixtures or DNA fragments.
• After a separation is completed the support is stained to visualize
the separated components.
• Resolution can be greatly improved using isoelectric focusing. In
this technique the support gel maintains a pH gradient. As a protein
migrates down the gel, it reaches a pH that is equal to its isoelectric
point. At this pH the protein is natural and no longer migrates, i.e, it
is focused into a sharp band on the gel.
Capillary Electrophoresis
Schematic of capillary electrophoresis
How Does CE Work?
• Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m
in length. The applied potential is 20 to 30 kV.
•Due to electro osmotic flow, all sample components migrate
towards the negative electrode. A small volume of sample (10 nL)
is injected at the positive end of the capillary and the separated
components are detected near the negative end of the capillary.
•CE detection is similar to detectors in HPLC and include
absorbance,
fluorescence,
electrochemical,
and
mass
spectrometry.
•The capillary can also be filled with a gel, which eliminates the
electro osmotic flow. Separation is accomplished as in
conventional gel electrophoresis but the capillary allows higher
resolution, greater sensitivity, and on-line detection.
well
Sample
(blue)
Gel Electrophoresis
(sodium dodecyl sulfate
polyacrylamide gel electrophoresis)
Is a vertical gel electropheresis
To separate proteins according to
their size
How Does GE Work?
•Electrophoresis refers to the electromotive force (EMF) that is used
to move the molecules through the gel matrix.
•By placing the molecules in wells in the gel and applying an electric
field, the molecules will move through the matrix at different rates,
determined largely by their mass
•Molecules move toward the (negatively charged) cathode if positively
charged or toward the (positively charged) anode if negatively
charged.
• Shorter molecules move faster and migrate farther than
longer ones because shorter molecules migrate more easily
through the pores of the gel.
•
•This phenomenon is called sieving.
Gel staining
 immersed in solution e.g ethidium bromide
Result
Sample 1
Sample 2
Sample 3
Bigger protein
reference
Smaller protein
marker