CH 172:

CHEMICAL SEPARATION

Liquid Chromatography
 LIQUID CHROMATOGRAPHY
(LC)
 Chromatography in which the mobile phase is a liquid.
 The liquid used as the mobile phase is called the
“eluent”.
 The stationary phase is usually a solid or a liquid.
 In general, it is possible to analyze any substance that
can be stably dissolved in the mobile phase.
 Interaction Between Solutes, Stationary Phase,
and Mobile Phase: Differences in the interactions
between the solutes and stationary and mobile
phases (Degree of adsorption, solubility, ionicity, etc.)
enable separation.

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From Liquid Chromatography to High
Performance Liquid Chromatography (HPLC)

 Higher degree of separation

 Refinement of packing material (3 to 10
µm)
 Reduction of analysis time!
 Delivery of eluent by pump
 Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid
chromatography

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 HPLC
 High Performance Liquid Chromatography
 High Pressure Liquid Chromatography
 High Priced Liquid Chromatography
 Chromatography = Analytical technique
 Chromatograph = Instrument
 Chromatogram = Obtained “picture”
 Chromatographer = Person
Partitioning
 Separation is based on the analyte’s relative
solubility between two liquid phases
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Instrumentation

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 Mobile Phase
 Water  Organic Solvent
 “Ultrapure water” can  HPLC-grade solvent can
be used with be used with confidence.
confidence.  Special-grade solvent is
 Commercial “distilled acceptable depending on
water for HPLC” is also the detection conditions.
acceptable.  Care is required
regarding solvents
containing stabilizers
(e.g., tetrahydrofuran
and chloroform)

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Buffer solutions used for HPLC eluent

 Requirements
 High buffering power at prescribed pH.
 Does not adversely affect detection.
 Does not damage column or equipment.
 Inexpensive.
 Commonly used acids:
 Phosphoric acid pKa 2.1, 7.2, 12.3
 Acetic acid pKa 4.8
 Citric acid pKa 3.1, 4.8, 6.4
 Concentration: If only to adjust pH, 10 mmol/L is
sufficient.
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Solvent Delivery
 Isocratic system
 Constant eluent composition
 Gradient system
 Varying eluent composition
 HPGE (High Pressure Gradient)
 LPGE (Low Pressure Gradient)
 Improves separation

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 HPLC Columns
 Solid Support - Backbone for bonded phases.
 Usually 10 µ, 5 µ or 3 µ silica or polymeric particles.
 Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
 Extremely stable
 Reproducible
 Guard - Protects the analytical column:
 Particles
 Interferences
 Prolongs the life of the analytical column
 Analytical-Performs the separation

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 HPLC Columns: Bonded Phases
 C-2 Ethyl Silyl -Si-CH2-CH3
 C-8 Octyl Silyl -Si-(CH2)7-CH3
 C-18 Octadecyl Silyl -Si-(CH2)17-CH3
 CN Cyanopropyl Silyl -Si-(CH2)3-CN

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HPLC Separation Modes
 Adsorption (liquid-solid) chromatography
 Partition (liquid-liquid) chromatography
 Normal phase partition chromatography
 Reversed phase partition chromatography
 Ion exchange chromatography
 Size exclusion chromatography

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 HPLC Separation Modes
Adsorption Chromatography
 A solid such as silica gel is used as the stationary phase,
and differences, mainly in the degree of adsorption to its
surface, are used to separate the solutes.
 Liquid-solid chromatography
 The retention strength increases with the hydrophilicity of
the solute.
Partition Chromatography
 A liquid (or a substance regarded as a liquid) is used as
the stationary phase, and the solute is separated
according to whether it dissolves more readily in the
stationary or mobile phase.
 Liquid-liquid chromatography
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 HPLC–Modes:
Normal Phase / Reversed Phase
Phase Stationary phase Mobile phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)
Reversed Low polarity High polarity
phase (hydrophobic) (hydrophilic)

Normal Phase: Polar stationary phase and non-polar

solvent.
Reverse Phase: Non-polar stationary phase and a polar
solvent.
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 Normal Phase (Partition)
Chromatography
 Partition chromatography in which the stationary
phase has a high polarity (hydrophilic) and the
mobile phase has a low polarity (hydrophobic)
 Essentially based on the same separation
mechanism as adsorption chromatography in
which the stationary phase has a hydrophilic
base, such as silica gel

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 Stationary phase and mobile phase
used in normal phase mode
 Stationary Phase
 Silica gel: -Si-OH
 Cyano type: -Si-CH2CH2CH2CN
 Amino type: -Si-CH2CH2CH2NH2
 Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
 Mobile Phase
 Basic solvents: Aliphatic hydrocarbons,
aromatic hydrocarbons, etc.
 Additional solvents: Alcohols, ethers, etc.

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Reversed Phase Chromatography
 Stationary phase: Low polarity
Separation Columns for Reversed Phase Chromatography:
 C18 (ODS) type = Octadecyl group-bonded silical gel (ODS),

 C8 (octyl) type, C4 (butyl) type, Phenyl type,

TMS type, and Cyano type
 Mobile phase: High polarity
 Common reverse phase solvents are: Methanol (CH3OH),
acetonitrile (CH3CN), water (H2O) and tetrahydrofuran

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Comparison of Normal Phase and
Reversed Phase
 Normal Phase  Reversed Phase
 Effective for separation  Wide range of
of structural isomers applications
 Offers separation  Effective for separation
selectivity not available of homologs
with reversed phase  Stationary phase has
 Stabilizes slowly and is long service life
prone to fluctuations in  Stabilizes quickly
retention time  Eluents are
 Eluents are expensive inexpensive and easy
to use
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Ion Exchange Chromatography

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Stationary phase used in ion exchange
mode
 Base Material
 Resin is often used.
 Silica gel is also used.
 Cation Exchange Column
 Strong cation exchange (SCX) -SO3-
 Week cation exchange (WCX) -COO-
 Anion Exchange Column
 Strong anion exchange (SAX) -NR3+
 Week anion exchange (WAX) -NHR2+

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 Size Exclusion Chromatography
 Separation is based on the size (bulkiness) of
molecules.
 The name varies with the application fields:
 Size Exclusion Chromatography (SEC)
 Gel Permeation Chromatography (GPC)
 Chemical industry fields, synthetic polymers,
nonaqueous systems
 Gel Filtration Chromatography (GFC)
 Biochemical fields, biological macromolecules,
aqueous systems
 Principle of Size Exclusion Mode: The size of the
solute molecules determines whether or not they can
enter the pores.
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 Guidelines for selecting separation
mode: Required information
 Soluble solvent
 Molecular weight
 Structural formula and chemical properties: Do the
substances ionize? Is there UV absorption or fluorescence?
Is derivatization possible? etc.
 Reversed phase mode using an ODS column is the first
choice.
 Exceptions
 Large molecular weight (> 2,000)  Size exclusion
 Stereoisomers, positional isomersNormal phase/
adsorption
 Inorganic ions  Ion chromatography
 Sugars, amino acids, short-chain fatty acidsSpecial
column
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 Detectors:
Detection Condition Requirements
 Sensitivity
 The detector must have the appropriate level of
sensitivity.
 Selectivity
 The detector must be able to detect the target
substance without, if possible, detecting other
substances.
 Adaptability to separation conditions
 Operability, etc.

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 Detectors
 UV-Vis absorbance detector
 Single wavelength (filter)
 Variable wavelength (monochromator)
 Multiple wavelengths, e.g. Photodiode Array
Detectors (PDA)
 Fluorescence
 Electrochemical
 Mass Spectrometric (MS), e.g LC-MS-excellent
selectivity; Peaks can be identified with MS spectra
 Refractive index detector
 Electrical conductivity detector
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Advantages of High Performance
Liquid Chromatography
 High separation capacity, enabling the batch
analysis of multiple components
 Superior quantitative capability and reproducibility
 Moderate analytical conditions
 Unlike GC, the sample does not need to be vaporized.
 Generally high sensitivity
 Low sample consumption
 Easy preparative separation and purification of
samples

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 Fields in Which HPLC is Used
 Charged, polar, thermolabile, non-volatile, and high-
molecular-weight compounds are some of the
chemicals that can be analyzed by HPLC.
 The fields in which HPLC is used include:
• Biogenic substances, e.g. Sugars, lipids, nucleic acids,
amino acids, proteins, peptides, steroids, amines, etc.
• Medical products, e.g. Drugs, antibiotics, etc.
• Food products, e.g. Vitamins, food additives, sugars,
organic acids, amino acids, etc.
• Environmental samples, e.g. Inorganic ions, Hazardous
organic substances, etc.
• Organic industrial products, e.g. Synthetic polymers,
additives, surfactants, etc.
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