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Chromatography
Chromatography
Detector Signal
1 2
time or volume
In practice the Column is contained in a thermostatic oven.
(Why ?)
Oven
Detector
Column
Nitrogen
cylinder
Chromatogram of petrol
•Carboxymethyl(CM)cellulose(cation exchanger)
SIZE EXCLUTION/GEL FILTERATION/GEL
PERMEATION
•
Pump Column
Detector
Injector
Mobile Phases
HPLC - Modes
• Normal Phase.
- Polar stationary phase and non-polar
solvent.
• Reverse Phase.
- Non-polar stationary phase and a polar
solvent.
Common Reverse Phase Solvents
• Methanol CH3OH
• Acetonitrile CH3CN
• Tetrahydrofuran
• Water H2O
• Solid Support - Backbone for bonded phases.
– Usually 10µ, 5µ or 3µ silica or polymeric particles.
• Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
– Extremely stable
– Reproducible
• Guard - Protects the analytical column:
– Particles
– Interferences
– Prolongs the life of the analytical column
Bonding Type
• Monomeric - single-point attachment of bonded phase molecule
• Polymeric - multi-point attachment of bonded phase molecule
Carbon Load
• Amount of bonded phase attached to base material, expressed as %C
Endcapping
• “Capping” of exposed silanols with short hydrocarbon chains after the primary
bonding step
COLUMN DIMENSIONS
Effect on chromatography
Column Dimension
• Short (30-50mm) - short run times, low backpressure
• Long (250-300mm) - higher resolution, long run times
• Narrow ( 2.1mm) - higher detector sensitivity
• Wide (10-22mm) - high sample loading
PARTICLE SHAPE
Effect on chromatography
Spherical particles offer reduced back pressures and longer column life when using viscous mobile
phases like 50:50 MeOH:H2O.
PARTICLE SIZE
Effect on chromatography
Smaller particles offer higher efficiency, but also cause higher backpressure. Choose 3µm particles for
resolving complex, multi-component samples. Otherwise, choose 5 or 10µm packings.
SURFACE AREA
Effect on chromatography
High surface area generally provides greater retention, capacity and resolution for separating complex,
multi-component samples. Low surface area packings generally equilibrate quickly, especially
important in gradient analyses.
PORE SIZE
Effect on chromatography
Larger pores allow larger solute molecules to be retained longer through maximum exposure to the
surface area of the particles. Choose a pore size of 150Å or less for sample MW 2000. Choose
a pore size of 300Å or greater for sample MW > 2000.
BONDING TYPE
Effect on chromatography
Monomeric bonding offers increased mass transfer rates, higher column efficiency, and faster column
equilibration.
CH3 R
monomeric
OH + X Si (CH2)17 CH3 Si (CH2)17 CH3
bonding
CH3 R
OH CH3 O CH3
polymeric
+ X Si (CH2)17 CH3 Si
bonding
OH X O (CH2)17 CH3
Loadability
High Stabil
Stability at
High Samp
Suitable fo
High Resol
Consumpti
Goals
High Sensi
Fast Analy
Fast Eqilib
High Effici
Metho
Backpress
Low Mobil
Applicatio
(Good for
Extremes
>2000
Low
Particle Size
small (3µm) • •
medium (5µm) •
large (10µm) •
Column Length
short (30mm) • • • • •
medium (150mm) •
long (300mm) •
Column ID
narrow (2.1mm) • • medium (4.6mm) •
wide (22.5mm) •
Surface Area
low (200m2/g) • • •
high (300m2/g) • • •
Pore Size
small (60Å) • •
medium (100Å) •
large (300Å) •
Carbon Load
low (3%) •
medium (10%) •
high (20%) • • •
Bonding Type
monomeric • •
polymeric • • • •
Particle Shape
spherical • • • •
irregular •
• UV
Detectors
– Single wavelength (filter)
– Variable wavelength (monochromator)
– Multiple wavelengths (PDA)
• Fluorescence
• Mass Spectrometric
Monochromator