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Isolation and Purification of Isolation and Purification of Organic CompoundsOrganic Compounds

Dr. Ralph C. GatroneDr. Ralph C. GatroneDepartment of Chemistry and PhysicsDepartment of Chemistry and Physics

Virginia State UniversityVirginia State University

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ObjectivesObjectives

ExtractionExtraction RecrystallizationRecrystallization Melting and Boiling PointsMelting and Boiling Points DistillationDistillation SublimationSublimation ChromatographyChromatography

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ExtractionExtraction

Based upon relative solubility between two Based upon relative solubility between two immiscible solventsimmiscible solvents

Useful for:Useful for:– Removing interferencesRemoving interferences– Concentrating speciesConcentrating species– Obtaining measurable amounts of materialObtaining measurable amounts of material

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ExtractionExtraction

Separation of a component from a mixture Separation of a component from a mixture by means of a solventby means of a solvent

Separatory funnel and shaking two Separatory funnel and shaking two immiscible solventsimmiscible solvents

Desired component is more soluble in the Desired component is more soluble in the extracting solventextracting solvent

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Separatory FunnelSeparatory Funnel

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Distribution CoefficientDistribution Coefficient

Defined asDefined as

Quantitative description the relative solubilityQuantitative description the relative solubility Assumes ideal behaviorAssumes ideal behavior Solvent A has density greater than or less Solvent A has density greater than or less

than onethan one Solvent B has density equal to oneSolvent B has density equal to one

Kd = concentration of solute in solvent A

concentration of solute in solvent B

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Multiple ExtractionsMultiple Extractions

It is not always possible to remove a It is not always possible to remove a substance on single extractionsubstance on single extraction

Increase volume of solventIncrease volume of solvent Use multiple extractionsUse multiple extractions More efficient methodMore efficient method

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RecrystallizationRecrystallization

Separation of a solid compound from Separation of a solid compound from impurities by differences in solubilitiesimpurities by differences in solubilities

Solubility varies with temperatureSolubility varies with temperature Majority of compounds have greater Majority of compounds have greater

solubility in hot solvents than coldsolubility in hot solvents than cold Critical aspect is choice of solventCritical aspect is choice of solvent Generally a trial and error processGenerally a trial and error process

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Solvent PropertiesSolvent Properties

Polarity – like dissolves likePolarity – like dissolves like High dielectric constants dissolve more High dielectric constants dissolve more

polar compoundspolar compounds (the dielectric constant is a relative measure (the dielectric constant is a relative measure

of how polar a solvent is –of how polar a solvent is –– Water: 80 at 20Water: 80 at 20oo C C– Hexane: 1.89 at 20Hexane: 1.89 at 20oo C C

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Melting and Boiling PointsMelting and Boiling Points Melting PointMelting Point Solids – finite vapor pressureSolids – finite vapor pressure As T increases the vapor pressure increasesAs T increases the vapor pressure increases At the mp – solid and liquid are at equilibriumAt the mp – solid and liquid are at equilibrium

solid liquid

vapor

melting

freezing

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Melting PointsMelting Points

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

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Boiling PointsBoiling Points

vapor pressure of liquid and gas phases are equalvapor pressure of liquid and gas phases are equal bp is dependent upon pressurebp is dependent upon pressure pressure and boiling point are recordedpressure and boiling point are recorded Water:Water: 100.3 degrees at -285’ (1.01atm)100.3 degrees at -285’ (1.01atm) 100.0 degrees at 0’ (1.00atm)100.0 degrees at 0’ (1.00atm) 93 degrees at 7520’ (0.75atm)93 degrees at 7520’ (0.75atm)

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Boiling PointsBoiling Points

Polar compounds have higher bp than non-polar Polar compounds have higher bp than non-polar compoundscompounds

Increasing MW increases bp (constant polarity)Increasing MW increases bp (constant polarity)

bp important for distillation to purify organic liquidsbp important for distillation to purify organic liquids

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DistillationDistillation

bp of mixtures dependent upon mole fraction of bp of mixtures dependent upon mole fraction of component presentcomponent 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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DistillationDistillation

SimpleSimple FractionalFractional VacuumVacuum SteamSteam

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Simple DistillationSimple Distillation

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Fractional DistillationFractional Distillation

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Which?Which?

SimpleSimple FractionalFractional Simple setupSimple setup Complicated setupComplicated setup Fast processFast process Slow processSlow process Consumes less EConsumes less E Energy intensiveEnergy intensive Poorer separationPoorer separation Better separationBetter separation

Best for relatively Best for relatively Best for mixtures Best for mixtures pure liquidspure liquids with close bpwith close bp

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AzeotropesAzeotropes

Constant boiling liquid mixturesConstant boiling liquid mixtures Cannot be purified further by distillationCannot be purified further by distillation 95.6% EtOH + 4.4% HOH: bp = 78.2 95.6% EtOH + 4.4% HOH: bp = 78.2 oo

Vapor composition is the same as the liquid Vapor composition is the same as the liquid compositioncomposition

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Vacuum DistillationVacuum Distillation

Boiling point is dependent upon pressureBoiling point is dependent upon pressure As pressure is reduced the bp reducesAs pressure is reduced the bp reduces Can distill high boiling organics by reducing Can distill high boiling organics by reducing

the pressure - vacuum distillationthe pressure - vacuum distillation

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Vacuum DistillationVacuum Distillation

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Vacuum PumpVacuum Pump

Oil Lubrica

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Steam DistillationSteam Distillation

co-distillation with waterco-distillation with water two components are immiscibletwo components are immiscible each exerts separate full vapor pressureeach exerts separate full vapor pressure total vapor pressure = total vapor pressuretotal vapor pressure = total vapor pressure T is always less than bp of waterT is always less than bp of water application in flavor and fragrance industriesapplication in flavor and fragrance industries

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SublimationSublimation

Evaporation generally requires meltingEvaporation generally requires melting Some substances evaporate from solid stateSome substances evaporate from solid state SublimationSublimation Iodine, carbon dioxideIodine, carbon dioxide High vapor pressures below mpHigh vapor pressures below mp

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Purification by SublimationPurification by Sublimation

Vaporize without meltingVaporize without melting Vaporizes without decompositionVaporizes without decomposition Vapor condenses to solidVapor condenses to solid Impurities present do not sublimeImpurities present do not sublime

Generally utilize reduced pressureGenerally utilize reduced pressure

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SublimationSublimation

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ChromatographyChromatography

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

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ChromatographyChromatography

Developed in early 1900’sDeveloped in early 1900’s Mikhail Semenovich TsvetMikhail Semenovich Tsvet Distribution of a substance between two phasesDistribution of a substance between two phases Stationary phaseStationary phase Mobile phaseMobile phase Affinity for stationary phase versus Affinity for stationary phase versus Solubility in mobile phaseSolubility in mobile phase Adsorption onto stationary phaseAdsorption onto stationary phase Desorption into mobile phaseDesorption into mobile phase Equilibrium process – partitions between two phasesEquilibrium process – partitions between two phases

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Thin-Layer ChromatographyThin-Layer Chromatography

Developed in late 1950’sDeveloped in late 1950’s

Simple, inexpensive, fast, efficient, sensitive, and requires Simple, inexpensive, fast, efficient, sensitive, and requires mg quantitiesmg quantities

Most useful forMost useful for Determining the number of componentsDetermining the number of components

Establishing whether two components are the sameEstablishing whether two components are the same

Following a reaction’s progressFollowing a reaction’s progress

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TLCTLC

Stationary phaseStationary phase glass or plastic plates coated with thin layer glass or plastic plates coated with thin layer

of adsorbentof adsorbent Silica gel, alumina, celluloseSilica gel, alumina, cellulose Mobile phaseMobile phase Solvent or mixture of solventsSolvent or mixture of solvents Determined by sample polarityDetermined by sample polarity

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Gas-Liquid ChromatographyGas-Liquid Chromatography

Analysis of volatile organic liquidsAnalysis of volatile organic liquids Quick and easy methodQuick and easy method QualitativeQualitative QuantitativeQuantitative Separates very complex mixturesSeparates very complex mixtures Compounds must have high vapor pressureCompounds must have high vapor pressure Known samples must be available for Known samples must be available for

identificationidentification

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GCGC

1952 by A. Martin and R. Synge1952 by A. Martin and R. Synge Stationary PhaseStationary Phase Non-volative liquidNon-volative liquid Packed column – coated on solid supportPacked column – coated on solid support Capillary column – thin film coated on Capillary column – thin film coated on

capillary tubecapillary tube Mobile phaseMobile phase Inert gas (He or NInert gas (He or N22))

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ProcessProcess

Sample is injectedSample is injected Heated injection portHeated injection port Vaporized into gasVaporized into gas Components are partitioned between gas Components are partitioned between gas

and stationary phaseand stationary phase Equilibrium depends uponEquilibrium depends upon Temperature, gas flow rate, solubility in Temperature, gas flow rate, solubility in

stationary phasestationary phase

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ColumnColumn

Packed ColumnsPacked Columns Interior diameter = 2 – 4 mmInterior diameter = 2 – 4 mm Length = 2 – 3 mLength = 2 – 3 m Coating = 0.05 – 1 micrometerCoating = 0.05 – 1 micrometer Capillary ColumnsCapillary Columns Interior diameter = 0.25 – 0.5 mmInterior diameter = 0.25 – 0.5 mm Length = 10 – 100 mLength = 10 – 100 m Coating = 0.1 – 5 micrometerCoating = 0.1 – 5 micrometer

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Stationary PhaseStationary Phase

Liquid phase is most efficient when it is Liquid phase is most efficient when it is similar to the material being separatedsimilar to the material being separated

Non-polar phases for non-polar compoundsNon-polar phases for non-polar compounds Polar phases for polar compoundsPolar phases for polar compounds Most be cognizant of temperature rangeMost be cognizant of temperature range Many types availableMany types available

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DetectorsDetectors

Senses material presentSenses material present Converts into electrical signalConverts into electrical signal Thermal conductivityThermal conductivity Flame ionizationFlame ionization Mass selectiveMass selective

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Thermal ConductivityThermal Conductivity

Heat loss is related to gas compositionHeat loss is related to gas composition Hot filament generates electrical signalHot filament generates electrical signal Constant in flow of He gasConstant in flow of He gas Sample causes change in electrical signalSample causes change in electrical signal

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Flame IonizationFlame Ionization

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

signalsignal

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FIDFID

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Liquid ChromatographyLiquid Chromatography

ColumnColumn FlashFlash High PerformanceHigh Performance Separate mixtures of low volatilitySeparate mixtures of low volatility Useful for nanogram to multi gram quantitiesUseful for nanogram to multi gram quantities

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Column ChromatographyColumn Chromatography

Vertical glass columnVertical glass column Stationary phaseStationary phase

– Silica gelSilica gel– AluminaAlumina– Reverse phaseReverse phase

Elution solventsElution solvents– Generally made progressively more polarGenerally made progressively more polar

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Flash ChromatographyFlash Chromatography

Gravity elution is time consumingGravity elution is time consuming Gas pressure is applied to push eluent Gas pressure is applied to push eluent

through columnthrough column Silica gel of much smaller pore size is usedSilica gel of much smaller pore size is used More efficient separations are obtainedMore efficient separations are obtained Gas pressure controls eluent flow rateGas pressure controls eluent flow rate

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HPLCHPLC

Faster more efficient separationsFaster more efficient separations Stationary phases – 3 – 10 micronsStationary phases – 3 – 10 microns Increased surface areaIncreased surface area Enhanced separation and sensitivityEnhanced separation and sensitivity Flow restrictions are managed using Flow restrictions are managed using

pressures of 1000 – 6000 psipressures of 1000 – 6000 psi Flows of 1 – 2 mL per minuteFlows of 1 – 2 mL per minute

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HPLC DetectorsHPLC Detectors

UV DetectorsUV Detectors– Fixed wavelengthFixed wavelength– Multi-wavelengthMulti-wavelength– Diode ArrayDiode Array

Electrochemical conductivityElectrochemical conductivity FluorescenceFluorescence Refractive indexRefractive index

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Refractive IndexRefractive Index

Bulk propertyBulk property Changes in Rf by solute in the eluentChanges in Rf by solute in the eluent Developed in 1942Developed in 1942 Limited sensitivityLimited sensitivity Useful for compounds thatUseful for compounds that

– Do not fluoresceDo not fluoresce– Do not absorb uvDo not absorb uv

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Rf SchematicRf Schematic

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FluorescenceFluorescence

Light is emitted by molecule excited by Light is emitted by molecule excited by electromagnetic radiationelectromagnetic radiation

PhotoluminescencePhotoluminescence Release of light stops on removal of sourceRelease of light stops on removal of source Release of light is immediateRelease of light is immediate FluorescentFluorescent Release is delayedRelease is delayed Release continues after removal of sourceRelease continues after removal of source PhosphorescentPhosphorescent

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FluorescenceFluorescence

Greater sensitivity to sample concentrationGreater sensitivity to sample concentration Lesser sensitivity to instrument instabilityLesser sensitivity to instrument instability

– Measured against low light backgroundMeasured against low light background

Very few compounds fluoresceVery few compounds fluoresce Primarily compounds from food, drugs, and Primarily compounds from food, drugs, and

dyes have this propertydyes have this property

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SchematicSchematic

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UV DetectorsUV Detectors

Compounds respond to light in 180 – 350 nmCompounds respond to light in 180 – 350 nm Contains pi electrons, lone pairs of electrons, Contains pi electrons, lone pairs of electrons,

carbonyls, etc.carbonyls, etc. Very sensitiveVery sensitive Relationship based upon Beer’s LawRelationship based upon Beer’s Law Fixed – single wavelength lamp; Hg at 254nmFixed – single wavelength lamp; Hg at 254nm

– InexpensiveInexpensive

– Somewhat sensitiveSomewhat sensitive

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Schematic of Fixed WavelengthSchematic of Fixed Wavelength

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UV DetectorsUV Detectors

Multi-wavelength DetectorMulti-wavelength Detector Light source releases light over a range of Light source releases light over a range of

wavelengthswavelengths Deuterium or Xenon lamps are usedDeuterium or Xenon lamps are used

– Dispersion and diode arrayDispersion and diode array– Dispersion detectors are almost not soldDispersion detectors are almost not sold– Diode array is most commonDiode array is most common

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Dispersion UV DetectorsDispersion UV Detectors

Light is dispersed before it enters cellLight is dispersed before it enters cell Fluorescent compounds disrupt detectionFluorescent compounds disrupt detection Generally not a problem, but must be Generally not a problem, but must be

consideredconsidered Response is a function the intensity of the Response is a function the intensity of the

transmitted lighttransmitted light

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Schematic of Dispersive CellSchematic of Dispersive Cell

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Diode ArrayDiode Array

Deuterium lampDeuterium lamp Light from all wavelengths is passed through the Light from all wavelengths is passed through the

cell and dispersed over an array of diodescell and dispersed over an array of diodes Light is continuously monitored by all diodesLight is continuously monitored by all diodes Fluorescence is still a concernFluorescence is still a concern Output from any diode may be looked atOutput from any diode may be looked at Sensitivity is a little less than fixed wavelengthSensitivity is a little less than fixed wavelength More than adequateMore than adequate

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Schematic of Diode ArraySchematic of Diode Array