ANCH111: ANALYTICAL CHEMISTRY (QUALITATIVE AND QUANTITATIVE)

TOPIC: CHROMATOGRAPHY
2nd SEMESTER | S.Y 2022-2023
LECTURER: Ma’am Jericha Carina Pascua, MSPH
TOPIC CHROMATOGRAPHY
SUBTOPIC
SUB SUBTOPIC

CHROMATOGRAPHY
• Is a technique used to separate and identify the
components of a mixture.
• Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.
• Molecules that spend most of their time in the
mobile phase are carried along faster.
• Chromatography is a laboratory technique that
separates components within a mixture by using
the differential affinities of the components for a
mobile medium and for a stationary adsorbing
medium through which they pass.

TERMINOLOGY
• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the
components (mobile phase)
• Stationary Medium – the part of the apparatus that • We can use chromatography to separate the
does not move with the sample (stationary phase) components of inks and dyes, such as those found
in pens, markers, clothing, and even candy shells.
Chromatography can also be used to separate the
colored pigments in plants.

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 RF (RETARDATION FACTOR) attraction to itself is stronger than the force of
gravity.
• Because molecules in mixtures have different
• Solubility – the degree to which a material (solute)
characteristics (such as size and solubility), they
dissolves into a solvent. Solutes dissolve into
travel at different speeds when pulled along a piece
solvents that have similar properties. (Like
of paper by a solvent (water).
dissolves like) This allows different solutes to be
• For example, black ink contains several colors.
separated by different combinations of solvents.
When the water flows through a word written in
• Separation of components depends on both their
black, the molecules of each one of the colors
solubility in the mobile phase and their differential
behave differently, resulting in a sort of “rainbow”
affinity to the mobile phase and the stationary
effect with the colors separating out at different
phase.
rates.
• We can determine the RF (or retardation factor) by
measuring the distance the pigments traveled up
THIN LAYER CHROMATOGRAPHY
the strip and the distance the water traveled. You
will need to calculate the RF for each of the colors • uses an absorbent material on flat glass or plastic
that are present in a sample. plates.
• It is used to detect pesticide or insecticide residues
in food.
• Thin-layer chromatography is also used in forensics
to analyze the dye composition of fibers.
• For substances that are very soluble in the liquid Rf
• Most samples are not
will be close to 1
colored and need to be
• For substances that are rather insoluble in the liquid
visualized with a UV lamp.
Rf will be close to 0

Here are two proper sized

TYPES OF CHROMATOGRAPHY spots, viewed under a UV lamp
PAPER CHROMATOGRAPHY
• Separates dried liquid samples with a liquid solvent
(mobile phase) and a paper strip (stationary phase)
GAS CHROMATOGRAPHY
• is used in airports to detect bombs and is used in
forensics in many different ways.
• It is used to analyze fibers on a persons body and
also analyze blood found at a crime scene.
• Helium is used to move a gaseous mixture through
a column of absorbent material.

PRINCIPLES OF PAPER CHROMATOGRAPHY

• Capillary Action – the movement of liquid within the
spaces of a porous material due to the forces of
adhesion, cohesion, and surface tension. The liquid
is able to move up the filter paper because its

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 • The solubility of the gases in the liquid phases
depends on a number of factors, but one of the
most important is the volatility of the liquid from
which the component vapor drives.
• Gases from low boiling liquids will spend less time
dissolved in the liquid phase, whereas gasses from
less volatile liquids will spend more time in solution
with the liquid phase.
• The less time a gas spends dissolved in the liquid
phases, the more time it spends flying along with
the carrier gas. Thus it moves through the
• A gas mixture, called the Mobile Phase, composed
stationary phase more quickly.
of an inert “carrier” gas (usually Helium) and various
other chemical components in the vapor phase
introduced at the Injection Port pass through a non-
moving material, called the... LIQUID CHROMATOGRAPHY
• Stationary Phase, usually tightly packed into a • A sample mixture is passed through a column
copper tube called the Column… packed with solid particles which may or may not be
- which separates the gas stream into its coated with another liquid.
particular components based on how “quickly” • With the proper solvents, packing conditions, some
they move through the stationary phase. The components in the sample will travel the column
components reach a... more slowly than others resulting in the desired
• Detector at different times, the detector generates separation.
an electrical signal which is sent to a... • is used in the world to test water samples to look
• Recorder to record the signal. for pollution in lakes and rivers.
• Since many chemical substances are solids or liquids • is used to analyze metal ions and organic
at room temperature, the stationary phase-mobile compounds in solutions.
phase must be heated to higher temperature in • uses liquids which may incorporate insoluble
order to “vaporize” the components of the mobile molecules.
phase. Thus an external heating device or...
• Oven is needed. The temperature of the oven need DIAGRAM OF SIMPLE LIQUID COLUMN
not be above the boiling points of the components CHROMATOGRAPHY
but rather above the “dew point” of the least
volatile component DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

Solvent(mobile or
moving phase)
OOOOOOOOOOO
A +B +C Sample
OOOOOOOOOOO
(A+B+C)
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOA OOOO
OOOOOOOOOO Column OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Solid Particles OOOOOOOOOO
OOOOOOOOOOO (packing material- OOOOO B OOOO
OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO

•
OOOOOOOOOOO OOOOO C OOOO
As the mobile phase passes through the stationary OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
phase, the different components of the mobile OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO

phase will dissolve into the liquid film of the OOOOOOOOOOO

OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO

stationary phase. Eluant (eluate)

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 TYPES OF LIQUID CHROMATOGRAPHY •
• Partitioning of the sample between 2 phases delays
• The 4 basic liquid chromatography modes are
or retains some components more than others to
named according to the mechanism involved:
effect separation.
1. Liquid/Solid Chromatography (adsorption
chromatography)
a. Normal Phase LSC ODPN(oxydipropionylnitrile)
b. Reverse Phase LSC
Normal Phase LLC
2. Liquid/Liquid Chromatography (partition
Reverse Phase LLC
chromatography)
a. Normal Phase LLC
b. Reverse Phase LLC
3. Ion Exchange Chromatography
NCCH3 CH2 OCH2 CH2 CN(Normal)
4. Gel Permeation Chromatography (exclusion CH3 (CH2 ) 16 CH3 (Reverse)
chromatography)

MOBILE PHASE LIQUID

ION-EXCHANGE CHROMATOGRAPHY
Liquid-Liquid Liquid-Solid
FORMAT Chromatography
(Partition)
Chromatography
(Adsorption) • Separation in Ion-exchange Chromatography is
based on the competition of different ionic
STATIONARY
Liquid Solid compounds of the sample for the active sites on the
PHASE
ion-exchange resin (column-packing).

Normal Phase Normal Phase Reverse Phase

Reverse Phase

Mobile Phase - Mobile Phase -

Nonpolar Polar
Stationary phase - Stationary phase -
Polar Nonpolar

Normal phase LS
Reverse phase LS

− + - +
SO3 Na
Si - O - H
  Silica Gel

GEL-PERMEATION CHROMATOGRAPHY

• The separation mechanism in LSC is based on the • Gel-Permeation Chromatography is a mechanical

competition of the components of the mixture sorting of molecules based on the size of the
sample for the active sites on an absorbent such as molecules in solution.
Silica Gel. • Small molecules are able to permeate more pores
and are, therefore, retained longer than large
LIQUID-LIQUID CHROMATOGRAPHY molecules.
• The stationary solid surface is coated with a 2nd
liquid (the Stationary Phase) which is immiscible in
the solvent (Mobile) phase.
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
• (or High pressure liquid chromatography, HPLC) is a
form of column chromatography used frequently in
biochemistry and analytical chemistry to separate,
identify, and quantify compounds. HPLC utilizes a
column that holds chromatographic packing
material (stationary phase), a pump that moves the
mobile phase(s) through the column, and a detector
that shows the retention times of the molecules.
Retention time varies depending on the interactions
between the stationary phase, molecules being
analyzed, and the solvent(s) used.

CHROMATOGRAPHY
• Normal Phase Chromatograaphy uses a polar
stationary phase and a non-polar mobile phase, and
works effectively for relatively polar analytes
• Reversed Phase Chromatography has a non-polar
stationary phase and an aqueous, moderately polar
mobile phase.
• RESULT

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