CHE029

MODULE 10– Analytical Chemistry • MOBILE PHASE

Lesson title: Separation Techniques (Chromatography) - Always composed of “liquid” or a “gaseous
component.”
• SEPARATED MOLECULES
Coverage: TYPES OF CHROMATOGRAPHY
• Chromatography • Column Chromatography (CC)
• Types of Chromatography • Adsorption Chromatography
• Partition Chromatography
• Paper Partition Chromatography (PC)
CHROMATOGRAPHY • Reverse Phase Chromatography (RPC)
- The process in which a solution of a mixture • Ion-Exchange Chromatography (IEC)
containing inert materials, drug principles, and • Molecular Exclusion Chromatography (MEC)
impurities is separated into its components while • Thin Layer Chromatography (TLC)
moving through a bed of fixed porous solid having • Gas Chromatography (GC)
different reversible affinities for the substances • High-Pressure Liquid Chromatography (HPLC)
separated
- Is a technique for separating mixtures into their COLUMN CHROMATOGRAPHY (CC)
components in order to analyze, identify, purify, - Simplest type
and/or quantify the mixture or components - Most common methods of protein purification
- An important biophysical technique that enables the - Also known as Elution Chromatography
separation, identification, and purification of the Mobile Phase (Eluant):
components of a mixture for qualitative and • Solvent (Product obtained is called Eluate)
quantitative analysis Stationary phase (Adsorbent):
- Useful means of separating and purifying complex • Purified Siliceous earth,
and closely related materials which are difficult to • Activated ammonia,
separate by classical methods based on differences in • Silica gel
solubility and volatility • Calcium carbonate (Wet or dry packed)
- Based on the principle where molecules in mixture
applied onto the surface or into the solid, and fluid
stationary phase (stable phase) is separating from
each other while moving with the aid of a mobile
phase

PRINCIPLES
1. Resolution of mixtures
2. Determination of homogeneity ADSORPTION CHROMATOGRAPHY
3. Comparison of substances suspected of being identical - Also known as Liquid-Solid Chromatography (LSC)
4. Purification MOBILE PHASE:
5. Concentration of substance from dilute solutions • Liquid or Gas
6. Identification and control of technical products STATIONARY PHASE:
7. Quantitative separation from complex mixtures • Adsorbent
8. Indication of molecular structure
- Separation of mixture through a competitive process
THREE BASIS OF THE CHROMATOGRAPHY TECHNIQUE in which the molecules of the mobile phase compete
• STATIONARY PHASE with analyte molecules for polar adsorption sites on
- Always composed of a “solid” phase or “a the adsorbent
layer of a liquid adsorbed on the surface a - The interaction between the mobile phase and
solid support”
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adsorbent sites are strong, only a small amount of between two immiscible liquids
analyte will be adsorbed at any time and the - In this process, the mobile phase, usually an organic
movement of the analyte through an adsorption solvent, moves slowly over the stationary phase,
column will be relatively rapid and closely follow the usually water, which is held in place by the fibers of
solvent front. the filter paper.
- Conversely, when interactions between the mobile - Different substances move over the paper at different
phase and the adsorbent are weak, greater amounts rates, depending upon the relative solubilities in the
of analyte will be adsorbed at a time and the immiscible solvents, resulting in a separation by
movement of the analyte through the column will be partition.
slow - Thus, if a solution of several different constituents is
ELUOTROPIC SCALE placed at one end of a piece of filter paper saturated
- The selection of the appropriate upper-phase liquid with water under equilibrium conditions in a closed
requires a polarity scale, –ranked according to their vessel, and an organic solvent is allowed to flow slowly
order of strength of adsorption through the paper, the different constituents will
move at different rates characteristic of each
PARTITION CHROMATOGRAPHY substance and separation occurs
- Also known as Liquid-Liquid Chromatography (LLC)
MOBILE PHASE: - The ratio of the distance traveled by the front of the
• Liquid mobile phase, from the point of application of the test
STATIONARY PHASE: substance, is designated as Rf value of the compound:
• Liquid 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
- Separation by LLC is based on Nernst’s law – two
practically immiscible solvents are in contact with - The ratio between the distances traveled by a given
each other and a substance which is soluble in each is compound and a reference substance constitutes the
added, the substance distributes itself in such a way Rr value. Both Rf and Rr values are characteristic
that at equilibrium and at a given temperature the constants for different compounds and may be used
ratio of the concentrations of the two solutions is a for identification purposes
constant THREE MAIN METHODS:
- It is the activity ratio rather than the concentration • Descending
ratio which remains constant. When the ratio • Ascending
concentration expresses a distribution value for a • Radial chromatography
single chemical species, the constant is designated a
partition coefficient or distribution coefficient (K) and REVERSE PHASE CHROMATOGRAPHY (RPC)
may be expressed mathematically as: - Based on Partition phenomenon
K= Cι/ Cμ MOBILE PHASE:
Where: • Polar solvent
- Cι represents the lower phase STATIONARY PHASE:
- Cμ represents the uppercase • Non polar solvent is fixed to the paper or solid column
- The equation may also be expressed in vice-versa material

PAPER PARTITION CHROMATOGRAPHY (PC) ION-EXCHANGE CHROMATOGRAPHY

MOBILE PHASE: MOBILE PHASE:
• Organic Solvent • Aqueous Salt Solutions
STATIONARY PHASE: • Crude sample containing charged molecules
• Water (held in place by the fibers of the filter paper) STATIONARY PHASE:
- The basic principle of separation is that the • Styrene-divinyl benzene polymer w/ covalently
differences in partition coefficients of substances attached ionic function
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- Materials used in columns are either cation or anion - White developed chromatogram is examined under
resins UV radiation
- Used in the separation of charged biomolecules BEST SOLVENT FOR EXPLORATORY RUN:
- Both contain exchangeable functional groups • Benzene with 10%ethanol
attached to an insoluble resinous matrix composed • Chloroform with 10%ethanol
of styrene-divinylbenzene polymer
- The crude sample containing charged molecules is - Colored products do not need special color
used as the mobile phase. When it passes through the producing reagent to detect the location of the spot
chromatographic column, molecules bind to - Colorless products are examined under UV radiation
oppositely charged sites in the stationary phase to make spts discernible
- Used in separation of proteins and protein synthesis UV SOURCES:
• Short wave (254nm)
MOLECULAR EXCLUSION CHROMATOGRAPHY • Long wave (360 nm)
- Also known as Gel filtration or Gel permeation
Chromatography ADVANTAGES OVER TLC:
MOBILE PHASE: • Requires short time (30 mins or less)
• Buffer that flows in between the matrix • Requires small amount of the material
STATIONARY PHASE: • Provides complete separation of components in
• Poroud Matrix complex mixtures
• Sensitive
- A separation procedure in which differential
migration of solute molecules is based on molecular GAS CHROMATOGRAPHY
size MOBILE PHASE:
- Column materials consist of network-like structure of • Inert Gas (Carrier Gas)
polymer materials having pores of various sizes which STATIONARY PHASE:
exclude some molecules but permit the passage of • Liquid substrate; consisting of a high-boiling
other smaller molecules liquid which is used to coat granular particles
The retention volume is expressed in: made of siliceous earth or firebrick
Vr = Vm + Kd x Vs - Utilized in separation of alcohol, esther, lipid, amino
Where: groups, and observation of enzymatic interactions
Kd = Distribution coefficient
Vr = Retention volume COLUMN
Vm = Total mobile phase volume - These coated particles are housed in a tube which is
Vs = Total stationary phase volume of the column made of copper, glass or stainless steel. It is mounted
in a constant-temperature chamber
THIN LAYER CHROMATOGRAPHY
MOBILE PHASE: HIGH PRESSURE LIQUID CHROMATOGRAPHY
• Developing solvent MOBILE PHASE:
STATIONARY PHASE: • Aqueous/Non-aqueous solvent
• A plate or strip coated with a form of Silica gel G - must be degassed to remove the formation of bubbles
(Gypsum as binder of the silica) (causes erroneous reading)
- Spotting of a sample of a mixture of components at STATIONARY PHASE:
one end of an adsorbent-coated glass plate or other • Silica
suitable support followed by the passage of a solvent • Powdered Chalk/Alumina
(developer) through the adsorbent for the purpose of
separating the components of a sample - Used to separate compounds that are dissolved
- Analysis is performed on a flat surface under - in solution
atmospheric pressure and room temperature - Separation using HPLC depend upon the processes of
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Adsorption, Partition, Ion-exchange, and Molecular

exclusion
- Greater speed, precision and accuracy, and general
ease in the operation of this chromatographic method
HPLC UTILIZES DETECTORS
• UV absorbance
• Refractive index
- Monitors the column effluent continuously as it
passes through the column, and a differential plot
(peak) is obtained, often using AUFS (absorbance
units full scale) on the chromatograms, with their
respective refractive index in one side of the reading
- It is used in laboratories to measure steroids,
barbiturates, and lipids

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