Chromatography
The use of chromatography in the organic lab
Agenda
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Introduction
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Very little theory
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Stationary phase
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Mobile phase
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Analytical methods
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Preparative methods
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Gravitational/Flash chromatography
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Dry Column Vacuum Chromatography
Theory
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Distribution coefficient
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Theoretical plates
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HETP
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Band broadening
Theory
The stationary phase
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Silica
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Aluminiumxoid
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Basic
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Acidic
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Activity
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E vs T
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Reversed-phase silica
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Cellulose, florisil, BioBeads, Sephadex
The stationary phase
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Silica Particle size
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63-200 μm - Gravitation/MPLC
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40-63 μm - Flash/MPLC
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15-40 μm - DCVC
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5-15 μm – TLC/HPLC
The mobile phase
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Not as restricted as the stationary phase
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Solvent parameters
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Polarity
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Selectivity
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Solvent groups
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Binary eluent mixtures
The mobile phase
Analytical methods
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Automated techniques
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Analytical HPLC
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LC-MS
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LC-NMR
TLC
TLC
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Aluminiumoxide - F254
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Reversed phase silica – No indicator!
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Silica - F254
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2D-TLC
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TLC-MS
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TLC-NMR
TLC
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Development / visualisation
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UV
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2,4-DNPH
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Ninhydrin
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CAN
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Many more!
Preparative methods
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Preparative TLC
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Easy transfer of results from analytical TLC
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Easy visualisation
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No need for further TLC-analysis
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Small amounts per plate
Column Chromatography
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HPLC
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Small particle size / High Pressure
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Very expensive columns
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Typically reverse phase
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Elaborate setup, DAD, fraction collector
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Great resolution
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Typically several runs needed for prep. scale
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Pre-columns and extensive wash needed
Column Chromatography
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Gravitational/classic column chromatography
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Slurry packed with a solvent reservoir
Typically done with 40-63μm or 63-200μm particle
size
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Good resolution possible on “known columns”
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Time consuming
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Often done “blindly”
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Can be done faster with pressure
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Huge amounts of solvent needed
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Eluent system and column length often guestimated
Column Chromatography
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Flash Column Chromatography
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Very narrow definition
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45-63μm particle size
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Defined flow rate: 5 cm / min
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Eluent mixture determined by TLC – Rf = 0.35
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Crude method - resolution only trivial ΔRf ≥ 0.15
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Optimum packed length = 18 cm
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1L solvent per gram loaded compound needed
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Amounts above 10 g are non trivial
Column Chromatography
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Dry Column Vacuum Chromatography
Column Chromatography
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Dry Column Vacuum Chromatography
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Equipment
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Silica – 15-40μm
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Solvents
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Length of packed column
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Loading amounts - 500 mg/cm² - π*r²
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Fraction Volume – Follows the column
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Packing
Column Chromatography
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Dry Column Vacuum Chromatography
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Scalability
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Economy
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Time saved in post column analysis and
evaporation
Separations impossible using Flash routinely
achievable
Shaping of gradient makes separation below TLC
resolution possible
Column Chromatography
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Dry Column Vacuum Chromatography
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Sample loading
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Gradient elution
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Choice of eluent system and gradient
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Example
25% EtOAc/Heptane give Rf = 0.3 with
large ΔRf
– 5 Steps until this polarity give a 5%
gradient (25/5)
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Other types of chromatography
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Size exclusion chromatography
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Chromatotron
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3D chromatotron
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Counter current
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Single run dry column
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Quartz columns
Modified silica
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Reverse phase
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Aromatics
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Doping
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Affinity Chromatography
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Chiral chromatography
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Self prepared modified silica