VIETNAM NATIONAL UNIVERSITY - HCMC
INTERNATIONAL UNIVERSITY
Instructor: Dr. Nguyễn Tấn Khôi
Subject: Analytical Chemistry
Group’s members
- Nguyễn Thị Mỹ Trang
- Phùng Đan Tâm
- Trần Lê Bảo Ngọc
- Huỳnh Quốc Thái
- Nguyễn Thị Thanh Vân
-Nguyễn Ngọc Trân
- Nguyễn Trương Quỳnh Nga
- Nguyễn Hữu Lộc
OUTLINE
I. Introduction
II. Classification
- Column and thin layer chromatography
- Gas-liquid chromatography
III. Chromatography for biochemistry
1. What is chromatography?
- Quality method that identify the components in mixture
by separate them
•Mobile phase : may be a liquid, a gas or a supercritical fluid
, that continuously flows through the column and carries the
analytes.
= solvent + the sample being separated
•Stationary phase :a solid or a liquid which is immobilized
on the support particles, or on the inner wall of the column
tubing.
= column packing material
2. The principle of chromatography
The physical characteristics of a molecule are exploited in
chromatography to affect a separation. (molecular mass,
boiling point, ionic strength or molecular size)
3. Purpose of Chromatography
Analytical: determine chemical composition of a sample
Preparative: purify and collect one or more components
of a sample
4. Chromatogram
 Graph showing detector response as a function of elution
time.
(tR)A
tM = dead time
tR = retention time
wB = peak width at base
tM
w
B
- Is the time between injection and the maximum of the detector
response for the analyte.
- Is dictated by:
Chemical
factors
Physics
-Stationary
phase
- Mobile phase
-Intermolecular
forces
-Temperature
Mobile phase
velocity
Column
dimension
RETENTION FACTOR
Rf = D1 / D2
*where
 D1 = distance that color traveled, measured from center of the
band of color to the point where the food color was applied
 D2 = total distance that solvent traveled
SCHEME
MOBILE
PHASE
GAS CHROMATOGRAPHY
(GC)
ATTRACTIVE
FORCE
LIQUID
CHROMATOGRAPHY
(LC)
ADSORTION
PARTITION
GAS – SOLID
CHROMATOGRAPHY
PAPER
CHROMATOGRAPHY
ION EXCHANGE
THIN LAYER
CHROMATOGRAPHY
(TLC)
GAS - LIQUID
CHROMATOGRAPHY
HIGH
PERFORMANCE
LIQUID
CHROMATOGRAPHY
(HPLC)
COLUMN
CHROMATOGRAPHY
(GRAVITY FORCE)
SIZE EXCLUSION
1.Base on the nature of adsorptive force
( the mechanism of separation)
1.1 Adsorption
1.2 Partition
1.3 Ion Exchange
1.4 Size Exclusion
2. Classification based on mobile phase:
Liquid – chromatography
Gas – chromatography
2.1 Liquid chromatography
 Liquid chromatography (LC) is a separation technique in
which the mobile phase is a liquid.
 Liquid chromatography can be carried out either in a
column or a plane.
Classification of liquid chromatography
High Performance liquid chromatography (HPLC)
Paper chromatography (PC)
Column chromatography (CC)
Thin layer chromatography (TLC)
Column chromatography
 The separation and purification
technique in a vertical glass
column.
The stationary phase is a
powdered absorbent
The mobile phase is a solvent
poured on top of column.
The analyzed mixture is loaded on
top of column.
Thin layer chromatography (TLC):
Thin layer chromatography is used to:
Monitor the progress of a reaction
Identify compounds in a given substance
Determine the purity of a substance
General
A stationary phase of a
thin layer of
adsorbent like silica
gel, alumina, or
cellulose on a flat,
inert substrate
Developing solution that
travels up the
stationary phase
Principle
 Components of samples separate phase depend on how much
they adsorb on the stationary phase and how much they dissolve
in the mobile phase.
Process
Clip
2.2 Gas – chromatography
• Is based upon a solid stationary
Gas-solid
(adsorption)
chromatography
phase on which retention of
analyte is the consequence of
physical adsorption
Gas-liquid
(partition)
chromatography
• Is based on the relative solubility
of analyte in mobile and
stationary phases
Gas chromatography
 A common type of chromatography used in analytic chemistry
for separating and analyzing
 Widely used for the determination of organic compounds that
can be vaporized without decomposition
Gas chromatographic
Gas-Liquid chromatographic
the stationary phase is an involatile liquid held on
particles of a solid support.
The mechanism
-Firstly, the process is carried out between a liquid stationary
phase and a gas moving phase
-Secondly, the various component of analytes are separated as
they progress
-Thirdly, detector is used to monitor the outlet stream from the
column
MASS SPECTROMETRY
INTRODUCTION
I. INTRODUCTION
Different elements can be uniquely identified by their mass
I. INTRODUCTION
I. INTRODUCTION
Mass spectrometry is an analytical tool used for
measuring the molecular mass of a sample
I. INTRODUCTION
Biotechnology
Pharmaceutical
Clinical
Geological
II. MS PRINCIPLES
II. MS PRINCIPLES
Mass-to-charge (m/z) ratio:
 The unit of mass indicated as the Dalton:
1Da= 1/12 of mass of single atom C , C = 12 mass units
 m/z : An abbreviation is used to indicate physical quantity by
diving the mass of an ion in Dalton into the number of charges
carried by the ion.
 A mass spectrometer measure the m/z ratio of ion created from
the molecules.
MS PROCEDURE
4 stages:
IONIZATION
IONISATION
ACCELERATION
DEFLECTION
DETECTION
MS PROCEDURE
MS PROCEDURE
MS PROCEDURE
•The sample  ionisation
chamber.
•The electrically heated metal
coil gives off electrons which
are attracted to the electron
trap(+)
IONIZATION
MS PROCEDURE
ACCELERATION
The positive ions
are repelled away from
the positive ionisation
chamber
and pass through three slits
MS PROCEDURE
DEFLECTION
• Different ions are deflected by the magnetic field by different amounts.
• The amount of deflection depends on:
+ The mass of the ion
+ The charge on the ion
MS PROCEDURE
• Only ion stream B makes it right
through the machine to the ion
detector.
• When an ion hits the metal box,
its charge is neutralized by an
electron jumping from the metal
on to the ion
DETECTION
IV. INSTRUMENTS
MS INSTRUMENTS
MS instruments consist of three modules :
How MS is used
when coupled with
GC and LC
I. Gas chromatography-mass spectrometry
(GC/MS or GC-MS)
 A gas chromatograph: separate different compounds. This stream
of separated compounds is fed directly into the ion source.
 A metallic filament to which voltage is applied. This filament
emits electrons which ionize the compounds. The ions can then
further fragment, yielding predictable patterns.
 Intact ions and fragments pass into the mass spectrometer's
analyzer and are eventually detected.
A mass spectrometer (left) directly coupled to a gas chromatograph (right)
II.Liquid chromatography mass spectrometry
(LC/MS or LC-MS)
 Separates compounds chromatographically before they are
introduced to the ion source and mass spectrometer.
 The mobile phase is liquid, usually a mixture of water and organic
solvents (instead of gas), so the ions fragments cannot yield
predictable patterns.
 Most commonly, an electrospray ionization source is used in
LC/MS. There are also some newly developed ionization techniques
like laser spray.
Chromatography for biochemistry
Some of application of Chromatography
Toxicology is an area where chromatography is used.
Separating and identifying different drugs of abuse.
In sports medicine, any illegal drugs will be picked up using
chromatographic techniques. (eg Gas chromatography)
The area of toxicology involves testing for the
use of illegal substances, poisons and
alcohol.
Using samples from a suspect such as hair, a
toxicologist can confirm whether a person
has used illegal drugs weeks ago or only
yesterday.
*Marijuana, an indian
hemp plant often trafficked
illegally.
In sports medicine
One of the roles of the masculinising hormone testosterone is to increase
muscle size and strength. Taking extra testosterone, or taking a chemical that the
body can use to create extra testosterone, could therefore enhance an athlete's
performance. For this reason taking it is banned by the World Anti-Doping
Agency (WADA).
The problem is that it is not always easy to measure these two
substances, particularly as they are only present in urine at very low
concentrations.
So, a testing that makes use of liquid chromatography-tandem mass
spectrometry. This method has incredibly high sensitivity and increases the power
with which officials can search for both testosterone and epitestosterone in a
sample.
Applications of LC-MS/MS techniques
 food safety
 environment
 pharmaceutical
Applications in food safety
Applications in food safety using turbulent flow chromatography.
By injecting food samples directly into the LC/MS system, which
eliminates time-consuming and costly sample pre-paration steps,
food safety and quality laboratories can achieve significant
analytical improvements.
Turbulent flow chromatography technology also allows the broad
selection of stationary phases.
These features make the technology a versatile and important tool in
the food safety arena.
Applications in environment
LC-MS has become an invaluable technique for trace analysis
of polar compounds in aqueous samples of the environment
and in water treatment.
LC-MS is of particular importance due to the imetus it has
provided for research into the occurrence and fate of polar
contaminants, and of their even more polar transformation
products.
Applications in pharmaceutical
The application of LC-MS in
areas :
systematic toxicological
analysis drugs of abuse
therapeutic drug monitoring
doping in sport
pesticides
peptides and further potential
forensic applications.
Chromatography for biochemistry