Fall, 2008

Isolation and Purification of Organic Compounds

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

Fall, 2008

Separatory Funnel

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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 20 o C – Hexane: 1.89 at 20 o 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 Fall, 2008 solid melting freezing liquid 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 mole fraction A = moles 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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    Simple Fractional Vacuum Steam

Distillation

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Simple Distillation

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Fractional Distillation

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Which?

     Simple Simple setup Fast process Consumes less E Poorer separation Fractional Complicated setup Slow process Energy intensive Better separation  Best for relatively pure liquids Best for mixtures with close bp 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

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Vacuum Distillation

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Oil Lubrica

Vacuum Pump

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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 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

Fall, 2008

Sublimation

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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 mg quantities   Most useful for Determining the number of components  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

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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 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 N 2 ) 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

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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 Fall, 2008 36

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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 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

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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

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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 Fall, 2008 46

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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 Fall, 2008 48

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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

Fall, 2008

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 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

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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 Beer’s Law Fixed – single wavelength lamp; Hg at 254nm – Inexpensive – Somewhat sensitive Fall, 2008 57

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 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

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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 Fall, 2008 62

Fall, 2008

Schematic of Diode Array

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