LECTURE 4: CHROMATOGRAPHY
Content:
- Chromatographic separation
- classifying analytical separations; column chromatography, planar
chromatography
- gas chromatography; introduction, application
- hplc; introduction, application
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
•
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:
1) The stationary phase
2) 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.
Next presentation!(G12)
•
Elaborate the following procedures in using
gas chromatography
– Extraction
– Standard solution
– Calibration curve
– Peak area determination
Your references
1. Practical manual
2. Text book
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.
Assignment-Group
• Based in the given diagram, explain
functions of each part of the GC.
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.
• Sample Introduction
- Must consider three rules:
1. All constituents injected into GC must be
volatile.
2. The analyte must be present at an
appropriate concentration.
3. 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.
Liquid Chromatography
1. High performance Liquid
Chromaography
2. Size Exclusion
3. Ion Exchange
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
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.
Determination of analyte using HPLC.
Exercise:
Signal (arbitrary units)
The calibration curve of an external
standards
50000
y = 8403.7x
R2 = 1
40000
30000
20000
10000
0
50
100
150
standards (ppm)
200
250