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Isolation and Purification

Methode

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Metode Isolasi dan Pemurnian

Extraction
Recrystallization
Melting and Boiling Points
Distillation
Sublimation
Chromatography

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Extraction

Based upon relative solubility between two


immiscible solvents
Useful for:
Removing interferences
Concentrating species
Obtaining measurable amounts of material

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Extraction

Separation of a component from a mixture by


means of a solvent
Separatory funnel and shaking two immiscible
solvents
Desired component is more soluble in the
extracting solvent

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Separatory Funnel

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Distribution Coefficient
Defined as
concentration of solute in solvent A
Kd = concentration of solute in solvent B

Quantitative description the relative solubility


Assumes ideal behavior
Solvent A has density greater than or less than one
Solvent B has density equal to one

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Multiple Extractions

It is not always possible to remove a substance


on single extraction
Increase volume of solvent
Use multiple extractions
More efficient method

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Recrystallization

Separation of a solid compound from impurities by


differences in solubilities
Solubility varies with temperature
Majority of compounds have greater solubility in hot
solvents than cold
Critical aspect is choice of solvent
Generally a trial and error process

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Solvent Properties

Polarity like dissolves like


High dielectric constants dissolve more polar
compounds
(the dielectric constant is a relative measure of
how polar a solvent is
Water: 80 at 20o C
Hexane: 1.89 at 20o C

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Melting and Boiling Points
Melting Point
Solids finite vapor pressure
As T increases the vapor pressure increases
At the mp solid and liquid are at equilibrium

vapor

melting
solid liquid
freezing
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Melting Points

Physical characteristic
Generally reproducible
Presence of trace impurities depresses mp
Pure compounds melt over 0.5 to 2 degrees
Impure compounds have larger ranges

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Boiling Points

vapor pressure of liquid and gas phases are equal


bp is dependent upon pressure
pressure and boiling point are recorded
Water:
100.3 degrees at -285 (1.01atm)
100.0 degrees at 0 (1.00atm)
93 degrees at 7520 (0.75atm)

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Boiling Points

Polar compounds have higher bp than non-polar


compounds
Increasing MW increases bp (constant polarity)

bp important for distillation to purify organic


liquids

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Distillation

bp of mixtures dependent upon mole fraction of


component present

moles A
mole fraction A =
moles A + moles B

partial pressure A = (mole fraction A)(vapor pressure A)

vapor pressure = vapor pressure A + vapor pressure B ...


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Distillation

Simple
Fractional
Vacuum
Steam

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Simple Distillation

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Fractional Distillation

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Which?

Simple Fractional
Simple setup Complicated setup
Fast process Slow process
Consumes less E Energy intensive
Poorer separation Better separation

Best for relatively Best for mixtures pure


liquids with close bp

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Azeotropes
Constant boiling liquid mixtures
Cannot be purified further by distillation
95.6% EtOH + 4.4% HOH: bp = 78.2 o
Vapor composition is the same as the liquid
composition

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Vacuum Distillation

Boiling point is dependent upon pressure


As pressure is reduced the bp reduces
Can distill high boiling organics by reducing
the pressure - vacuum distillation

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Vacuum Distillation

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Vacuum Pump

Oil Lubricated Vacuum Pumps

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Steam Distillation

co-distillation with water


two components are immiscible
each exerts separate full vapor pressure
total vapor pressure = total vapor pressure
T is always less than bp of water
application in flavor and fragrance industries

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Sublimation
Evaporation generally requires melting
Some substances evaporate from solid state
Sublimation
Iodine, carbon dioxide
High vapor pressures below mp

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Purification by Sublimation

Vaporize without melting


Vaporizes without decomposition
Vapor condenses to solid
Impurities present do not sublime

Generally utilize reduced pressure

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Sublimation

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Chromatography

Thin-Layer (TLC)
Gas-Liquid (GC)
Liquid (LC)

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Chromatography

Developed in early 1900s


Mikhail Semenovich Tsvet
Distribution of a substance between two phases
Stationary phase
Mobile phase
Affinity for stationary phase versus
Solubility in mobile phase
Adsorption onto stationary phase
Desorption into mobile phase
Equilibrium process partitions between two phases
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Thin-Layer Chromatography
Developed in late 1950s
Simple, inexpensive, fast, efficient, sensitive, and
requires mg quantities
Most useful for
Determining the number of components
Establishing whether two components are the same
Following a reactions progress

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TLC

Stationary phase
glass or plastic plates coated with thin layer of
adsorbent
Silica gel, alumina, cellulose
Mobile phase
Solvent or mixture of solvents
Determined by sample polarity

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Gas-Liquid Chromatography

Analysis of volatile organic liquids


Quick and easy method
Qualitative
Quantitative
Separates very complex mixtures
Compounds must have high vapor pressure
Known samples must be available for identification

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GC

1952 by A. Martin and R. Synge


Stationary Phase
Non-volative liquid
Packed column coated on solid support
Capillary column thin film coated on capillary
tube
Mobile phase
Inert gas (He or N2)

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Process

Sample is injected
Heated injection port
Vaporized into gas
Components are partitioned between gas and
stationary phase
Equilibrium depends upon
Temperature, gas flow rate, solubility in stationary
phase

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Column
Packed Columns
Interior diameter = 2 4 mm
Length = 2 3 m
Coating = 0.05 1 micrometer
Capillary Columns
Interior diameter = 0.25 0.5 mm
Length = 10 100 m
Coating = 0.1 5 micrometer

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Stationary Phase

Liquid phase is most efficient when it is similar


to the material being separated
Non-polar phases for non-polar compounds
Polar phases for polar compounds
Most be cognizant of temperature range
Many types available

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Detectors

Senses material present


Converts into electrical signal
Thermal conductivity
Flame ionization
Mass selective

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Thermal Conductivity
Heat loss is related to gas composition
Hot filament generates electrical signal
Constant in flow of He gas
Sample causes change in electrical signal

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Flame Ionization

More sensitive
Non-flammable samples are not detected
Carrier gas is mixed with hydrogen
Sample is burned producing ions
These alter electrical output generating a signal

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FID

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Liquid Chromatography

Column
Flash
High Performance
Separate mixtures of low volatility
Useful for nanogram to multi gram quantities

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Column Chromatography

Vertical glass column


Stationary phase
Silica gel
Alumina
Reverse phase
Elution solvents
Generally made progressively more polar

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Flash Chromatography

Gravity elution is time consuming


Gas pressure is applied to push eluent through
column
Silica gel of much smaller pore size is used
More efficient separations are obtained
Gas pressure controls eluent flow rate

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HPLC

Faster more efficient separations


Stationary phases 3 10 microns
Increased surface area
Enhanced separation and sensitivity
Flow restrictions are managed using pressures
of 1000 6000 psi
Flows of 1 2 mL per minute

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HPLC Detectors

UV Detectors
Fixed wavelength
Multi-wavelength
Diode Array
Electrochemical conductivity
Fluorescence
Refractive index

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Refractive Index
Bulk property
Changes in Rf by solute in the eluent
Developed in 1942
Limited sensitivity
Useful for compounds that
Do not fluoresce
Do not absorb uv

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Rf Schematic

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Fluorescence
Light is emitted by molecule excited by
electromagnetic radiation
Photoluminescence
Release of light stops on removal of source
Release of light is immediate
Fluorescent
Release is delayed
Release continues after removal of source
Phosphorescent

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Fluorescence

Greater sensitivity to sample concentration


Lesser sensitivity to instrument instability
Measured against low light background
Very few compounds fluoresce
Primarily compounds from food, drugs, and
dyes have this property

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Schematic

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UV Detectors
Compounds respond to light in 180 350 nm
Contains pi electrons, lone pairs of electrons,
carbonyls, etc.
Very sensitive
Relationship based upon Beers Law
Fixed single wavelength lamp; Hg at 254nm
Inexpensive
Somewhat sensitive

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Schematic of Fixed Wavelength

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UV Detectors

Multi-wavelength Detector
Light source releases light over a range of
wavelengths
Deuterium or Xenon lamps are used
Dispersion and diode array
Dispersion detectors are almost not sold
Diode array is most common

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Dispersion UV Detectors

Light is dispersed before it enters cell


Fluorescent compounds disrupt detection
Generally not a problem, but must be
considered
Response is a function the intensity of the
transmitted light

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Schematic of Dispersive Cell

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Diode Array

Deuterium lamp
Light from all wavelengths is passed through
the cell and dispersed over an array of diodes
Light is continuously monitored by all diodes
Fluorescence is still a concern
Output from any diode may be looked at
Sensitivity is a little less than fixed wavelength
More than adequate

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Schematic of Diode Array

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