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