Isolation and Purification of

Organic Compounds
Dr. Ralph C. Gatrone
Department of Chemistry and Physics
Virginia State University

Fall, 2008 1
 Objectives
 Extraction
 Recrystallization
 Melting and Boiling Points
 Distillation
 Sublimation
 Chromatography

Fall, 2008 2
 Extraction
 Based upon relative solubility between two
immiscible solvents
 Useful for:
– Removing interferences
– Concentrating species
– Obtaining measurable amounts of material

Fall, 2008 3
 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

Fall, 2008 4
 Separatory Funnel

Fall, 2008 5
 Distribution Coefficient
 Defined as
Kd = concentration of solute in solvent A
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

Fall, 2008 6
 Multiple Extractions
 It is not always possible to remove a
substance on single extraction
 Increase volume of solvent
 Use multiple extractions
 More efficient method

Fall, 2008 7
 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

Fall, 2008 8
 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

Fall, 2008 9
 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

Fall, 2008 10
 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

Fall, 2008 11
 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)

Fall, 2008 12
 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

Fall, 2008 13
 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 ...

Fall, 2008 14
 Distillation
 Simple
 Fractional
 Vacuum
 Steam

Fall, 2008 15
 Simple Distillation

Fall, 2008 16
 Fractional Distillation

Fall, 2008 17
 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

Fall, 2008 18
 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

Fall, 2008 19
 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

Fall, 2008 20
 Vacuum Distillation

Fall, 2008 21
Oil Lubrica

Vacuum Pump

Fall, 2008 22
 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

Fall, 2008 23
 Sublimation
 Evaporation generally requires melting
 Some substances evaporate from solid state
 Sublimation
 Iodine, carbon dioxide
 High vapor pressures below mp

Fall, 2008 24
 Purification by Sublimation
 Vaporize without melting
 Vaporizes without decomposition
 Vapor condenses to solid
 Impurities present do not sublime

 Generally utilize reduced pressure

Fall, 2008 25
 Sublimation

Fall, 2008 26
 Chromatography
 Thin-Layer (TLC)
 Gas-Liquid (GC)
 Liquid (LC)

Fall, 2008 27
 Chromatography
 Developed in early 1900’s
 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

Fall, 2008 28
 Thin-Layer Chromatography
 Developed in late 1950’s

 Simple, inexpensive, fast, efficient, sensitive, and requires

 Most useful for

 Establishing whether two components are the same

 Following a reaction’s progress

Fall, 2008 29
 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

Fall, 2008 30
Fall, 2008 31
 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

Fall, 2008 32
 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)

Fall, 2008 33
 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

Fall, 2008 34
Fall, 2008 35
 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
Fall, 2008 36
Fall, 2008 37
 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

Fall, 2008 38
 Detectors
 Senses material present
 Converts into electrical signal
 Thermal conductivity
 Flame ionization
 Mass selective

Fall, 2008 39
 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

Fall, 2008 40
 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

Fall, 2008 41
 FID

Fall, 2008 42
 Liquid Chromatography
 Column
 Flash
 High Performance
 Separate mixtures of low volatility
 Useful for nanogram to multi gram quantities

Fall, 2008 43
 Column Chromatography
 Vertical glass column
 Stationary phase
– Silica gel
– Alumina
– Reverse phase
 Elution solvents
– Generally made progressively more polar

Fall, 2008 44
Fall, 2008 45
 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

Fall, 2008 46
Fall, 2008 47
 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

Fall, 2008 48
Fall, 2008 49
Fall, 2008 50
 HPLC Detectors
 UV Detectors
– Fixed wavelength
– Multi-wavelength
– Diode Array
 Electrochemical conductivity
 Fluorescence
 Refractive index

Fall, 2008 51
 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

Fall, 2008 52
 Rf Schematic

Fall, 2008 53
 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

Fall, 2008 54
 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

Fall, 2008 55
 Schematic

Fall, 2008 56
 UV Detectors
 Compounds respond to light in 180 – 350 nm
 Contains pi electrons, lone pairs of electrons,
carbonyls, etc.
 Very sensitive
 Relationship based upon Beer’s Law
 Fixed – single wavelength lamp; Hg at 254nm
– Inexpensive
– Somewhat sensitive

Fall, 2008 57
 Schematic of Fixed Wavelength

Fall, 2008 58
 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

Fall, 2008 59
 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

Fall, 2008 60
 Schematic of Dispersive Cell

Fall, 2008 61
 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

Fall, 2008 62
 Schematic of Diode Array

Fall, 2008 63