Fall, Isolation and Purification of Organic Compounds Dr. Ralph C. Gatrone Department of Chemistry and Physics Virginia State University

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

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

Fall, 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, Separatory Funnel

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

Fall, 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, 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, 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, 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

Fall, 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, Boiling Points  vapor pressure of liquid and gas phases are equal  bp is dependent upon pressure  pressure and boiling point are recorded  Water:  degrees at -285’ (1.01atm)  degrees at 0’ (1.00atm)  93 degrees at 7520’ (0.75atm)

Fall, 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, Distillation  bp of mixtures dependent upon mole fraction of component present

Fall, Distillation  Simple  Fractional  Vacuum  Steam

Fall, Simple Distillation

Fall, Fractional Distillation

Fall, Which?  SimpleFractional  Simple setupComplicated setup  Fast processSlow process  Consumes less EEnergy intensive  Poorer separationBetter separation  Best for relatively Best for mixtures pure liquidswith close bp

Fall, 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, 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, Vacuum Distillation

Fall, Vacuum Pump

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

Fall, Purification by Sublimation  Vaporize without melting  Vaporizes without decomposition  Vapor condenses to solid  Impurities present do not sublime  Generally utilize reduced pressure

Fall, Sublimation

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

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

Fall, 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, 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, FID

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

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

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

Fall, 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, Rf Schematic

Fall, 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, 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, Schematic

Fall, 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, Schematic of Fixed Wavelength

Fall, 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, 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, Schematic of Dispersive Cell

Fall, 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, Schematic of Diode Array