PROF. ANTONIO F.
LAUDE
CABRAL,GWYNETH
GAMBOA, FRANCHESCA
Techniques used to separate complex mixtures
or specimen compounds between mobile and
stationary phase
1. Mobile Phase - carries the complex mixture
2. Stationary phase - through which mobile phase
flows
3. Column - holds the stationary phase
4. Eluate - separated components
Use of Chromatography
1. Identification in serum or urine of drugs, sugars and
amino acids
2. Purification processes
3. Identification and quantitation of compounds
Mobile Phase, Stationary phase, Column and
Eluate
Mobile Phase, Stationary phase, Column and
Eluate
Modes of Separation
1. Adsorption
2. Partition
3. Steric Exclusion
4. Ion Exchange
Modes of Separation
1. Adsorption
Also known as
liquid-solid
chromatography
Based on the
competition
between the sample
and the solvent
(mobile phase) for
adsorptive sites on
the solid stationary
phase.
Plant pigments
extracted in hexane
Modes of Separation
1. Adsorption
Mixture is separated
into classes
Stationary phase
Acidic polar (silica
gel)
Basic polar (alumina)
Nonpolar (charcoal)
Example
Paper
Chromatography and
Thin-layer Chromato.
Plant pigments
extracted in hexane
Modes of Separation
2. Partition
Also known as liquid-liquid
chromatography
Separation of solute based on the relative
solubility of the compound in organic
(non-polar) and aqueous (polar) solvents
Polar molecules remain in the aqueous
solvent
Non polar molecules are extracted in the
organic solvent
Modes of Separation
3. Steric Exclusion
Also known as gel filtration, gel
permeation, size-exclusion, molecular
exclusion or molecular sieve
chromatography
Separate solute on the basis of size and
shape
Modes of Separation
3. Steric Exclusion
Modes of Separation
4. Ion Exchange
Solute mixtures are separated by magnitude
and charge of ionic species
Solute ions in the mobile phase exchange
with the opposite ions bound to the
stationary phase.
Stationary phase
Cation-exchange resin (side chains: H+
ions)
anion-exchange resin (side chains: OHions)
Modes of Separation
4. Ion Exchange
Chromatographic procedures
1. Planar Chromatography
a. Paper chromatography
b. Thin-layer chromatography
2. Column Chromatography
a. Gas chromatography
b. Liquid chromatography
Chromatographic procedures
1. Planar Chromatography
Stationary phase is coated on a sheet of paper or
bound to glass or plastic plate
Kinds of Planar Chromatography
a. Paper chromatography
b. Thin-layer chromatography
Chromatographic procedures
1. Planar Chromatography
a. Paper chromatography
The mixture to be
fractionated is place on
Whatman paper just
above solvent layer
The solvent move up
through the paper by
capillary action and the
fractions move up at
different rates
Chromatographic procedures
1. Planar Chromatography
b. Thin layer chromatography
The stationary phase is a
thin layer sorbent (i.e. silica
gel) coated on a glass plate
or a plastic sheet.
The mobile phase (solvent)
is place in one edge of the
plate
Samples are applied as
spots near one edge of the
plate
Chromatographic procedures
1. Planar Chromatography
b. Thin layer
chromatography
The solvent migrates up
by capillary action,
dissolving and carrying
sample molecules
Absorbance in each
developed spot is
measured by
densitometer.
Concentration is
calculated by comparison
with a reference standard
Chromatographic procedures
1. Planar Chromatography
b. Thin layer
chromatography
Rf
=
Distance travelled by
compounds from the
origin
Distance travelled
by solvent from the
origin
Chromatographic procedures
2. Column Chromatography
The stationary phase is packed into a tube or coated
onto the inner surface of the tube.
a. Liquid chromatography
b. Gas chromatography
Chromatographic procedures
2. Column Chromatography
a. Liquid chromatography
Separation is based on
the distribution of
solutes between a
liquid mobile phase
and stationary phase.
Chromatographic procedures
2. Column Chromatography
a. Liquid chromatography
i. High-performance
liquid chromatography
A high pressure
pump force the
solvent and sample
through a column
Chromatographic procedures
2. Column Chromatography
a. Liquid chromatography (HPLC)
i.
ii.
iii.
Pumps
Forces the mobile phase through the
column (i.e. pneumatic, syringe, etc.)
Columns
Stationary phase (i.e. silica gel)
Sample Injectors
introduce the sample into the mobile
phase
(i.e loop injector)
Chromatographic procedures
2. Column Chromatography
a. Liquid chromatography (HPLC)
iv. Detectors
produce an electronic signal proportional
to the concentration of each separated
component
photometer, flourometer, refractometer
Chromatographic procedures
2. Column Chromatography
a. Liquid chromatography (HPLC)
v. Recorders
Chromatogra
m
Chromatographic procedures
2. Column Chromatography
a. Liquid chromatography (HPLC)
Chromatogra
m
Recorde
r
Stationary
phase
Pumps
Chromatographic procedures
2. Column Chromatography
b. Gas chromatography
Used to separate mixture of compounds
that are volatile made or can be made
volatile
i. Gas-solid chromatography
uses a solid stationary phase
ii. Gas-liquid chromatography
uses a liquid coated on solid support
Chromatographic procedures
2. Column Chromatography
b. Gas chromatography
i.
ii.
iii.
Gas Cylinder (mobile phase)
Must be chemically inert. i.e. helium,
hydrogen ,etc.
Sample injector
Hypodermic syringe or automated sampler
Columns
Made of glass or stainless steel filled with
inert particles coated with a nonvolatile
liquid
(stationary phase)
Chromatographic procedures
2. Column Chromatography
b. Gas chromatography
iv. Detectors
Thermal conductivity (TC)
Flame ionization
most widely used and more sensitive
v. Recorders
Chromatographic procedures
2. Column Chromatography
b. Gas chromatography
Colum
n
Cylinder
of the
mobile
phase
Record
er
Syring
e
Detecto
r
Definition:
The process of separating the charged constituents
of a sample by means of an electrical current.
i.
Iontophoresis
Migration of small ions
ii.
Zone electrophoresis
Migration of charged macromolecules in a porous support
(paper. Cellulose acetate or agarose gel
Electrophoretogram
Result of electrophoresis consisting of separated
strands of a macromolecule
Agarose Gel Electrophoresis.
Components of Electrophoresis
i.
ii.
iii.
iv.
v.
Driving force (electrical power)
Support medium
Buffer
Sample
Detecting System
Components of Electrophoresis
Components of Electrophoresis
Components of Electrophoresis
Components of Electrophoresis
Detecting System (UV transillumination)
Components of Electrophoresis
Detecting System (Densitometer)
Components of Electrophoresis
Detecting System
Electrophoretogram
Result of electrophoresis consisting of separated
strands of a macromolecule
Agarose Gel Electrophoresis.
Components of Electrophoresis
i.
ii.
iii.
iv.
v.
Driving force (electrical power)
Support medium
Buffer
Sample
Detecting System
Charged particles migrate toward the
opposite charged electrode
Velocity of migration is controlled by:
i.
ii.
iii.
iv.
v.
net charge of the particle
Size and shape of the particle
Strength of the electric fields
Chemical and physical properties o the
supporting medium
Electrophoresis temperature
Power Supply
Buffers
Constant current or
voltage
If a protein is placed in a
solution that has a pH
higher that the pI, the
protein will bear a
negative charge
Whereas at a pH less
that the pI, the protein
will be positively
charged.
Power Supply
Buffers
Constant current or
voltage
If a protein is placed in a
solution that has a pH
higher that the pI, the
protein will bear a
negative charge
Whereas at a pH less
that the pI, the protein
will be positively
charged.
Support materials
i.
Cellulose acetate
Cellulose acetylated with acetic
anhydride
Separates serum proteins into 5
bands
ii. Agarose Gel
Purified fraction of agar
10 -15 bands
iii. Polyacrylamide gel
Separates proteins with more
fraction than cellulose
acetate/agarose (>20 bands)
Electroendosmosis
Isoelectric focusing
Capillary electrophoresis
Movement of buffer and solvent relative to their
fixed support.
Movement of buffer and solvent relative to their
fixed support.
Separation is performed in narrow-bore fuse silica
capillaries
Measures current or voltage (potential)
generated by the activity of specific ions in
analytes undergoing electrochemical
oxidative-reductive reactions
Electrode design involves two
linked electrochemical reactions
A. Reference electrode
Electrode with a constant voltage
B. Analytical electrode
Measuring electrode
Reference Electrode (half cells)
Serve as reference potential against unknown
voltage.
A. Silver-silver chloride
B. Calomel (Hg2Cl2)electrode
The voltage difference
between reference
electrode and the
analytical electrode can
be measured
Blood Gas Instruments
1. pH Electrode
2. pCO2 Electrode
3. pO2 Electrode
Blood Gas Instruments
1. pH Electrode
Measure hydrogen ion
activity
2. pCO2 Electrode
pH electrode with a
CO2-permeable membrane
and bicarbonate buffer.
Severinghaus electrode
Blood Gas Instruments
3. pO2 Electrode
Measures current flow
produced from loss or gain
of electrons.
The current flows as the
oxygen is reduced at the
cathode.
O2 + H2O + 2e- 2OHAg Ag+ + e- (Ag-AgCl
anode)
Clark electrode
Ion-Selective Electrodes
Composed of an
electrochemical half-cell and
an ion-specific
membrane
1. Sodium electrode
300 x sensitive than potassium
2. Potassium electrode with
valinomycin
1000x sensitive than sodium
3. Calcium electrode
Measurement of differences in voltage at a
constant current
Reference electrode; calomel and silver
chloride
Example. Measuring of pH and pressurized
CO2
Measurement of the amount of electricity in
coulombs at a fixed potential
Is an electrochemical titration in which the
titrant is electrochemically generated and the
endpoint is detected AMPEROMETRY.
Interferences; Bromide, Cyanide and Cysteine
Follows Faradays Law
