Gas Chromatography
Gas Chromatography
Gas Chromatography
Gas Chromatography
an analytical separations technique useful
Principles
Separation due to differences in partitioning
behavior
selective retardation
Key Information
Gas Chromatography
Separation column containing stationary phase
since partitioning behavior independent of
temperature - kept in thermostat - controlled oven
Detector
Schematic of a gas
Chromatograph
The Beginning
concept of GC announced in 1941 by
Today
estimate : 200, 000 gas chromatographs are
& liquids)
Determination of partition coefficients and
absorption isotherms
Isolating pure components from complex
mixtures
Instrumentation
Instrumentation
flowing mobile phase
injection port
separation column
detector
GC detectors
another powerpoint
Liquid Chromatography
much slower diffusion in liquid
as compared to gas
Elution chromatography
Increasing polarity of
Solvents mixed
pure solvents
hexane
ether
acetone
methanol
water
acetic acid
%hexane and %
methanol
miscible
can be mixed
continuously (solvent
programming)
What is HPLC?
The most widely used analytical separations technique
Utilizes a liquid mobile phase to separate components
of mixture
uses high pressure to push solvent through the column
Popularity:
sensitivity
ready adaptability to accurate quantitative determination
suitability for separating nonvolatile species or
thermally fragile ones
HPLC is.
Popularity:
widespread applicability to substances that are of prime
interest to industry, to many fields of science, and to the
public
Ideally suited for separation and identification of
History lesson
Early LC carried out in glass columns
diameters: 1-5 cm
lengths: 50-500 cm
Size of solid stationary phase
diameters: 150-200 m
Flow rates still low! Separation times long!
Eureka! Decrease particle size of packing causes increase in
column efficiency!
diameters 3-10 m
This technology required sophisticated instruments
new method called HPLC
Advantages to HPLC
Higher resolution and speed of analysis
HPLC columns can be reused without repacking or
regeneration
Greater reproducibility due to close control of the
parameters affecting the efficiency of separation
Easy automation of instrument operation and data
analysis
Adaptability to large-scale, preparative procedures
Advantages to HPLC
Advantages of HPLC are result of 2 major advances:
stationary supports with very small particle sizes and
large surface areas
appliance of high pressure to solvent flow
Schematic of liquid
chromatograph
LC column
LC injector
Types of HPLC
Liquid-solid (adsorption) chromatography
Liquid-liquid (partition) chromatography
Ion-exchange chromatography
Size exclusion chromatography
Partition Chromatography
Most widely used
Bonded-phase Chromatography
Silica Stationary Phase:
OH
OH
O
Si
Siloxanes:
O
Si
O
OH
Si
O
O
OH
O
Si
Si
CH 3
Si
R
CH3
R= C 8, C18
Partition Chromatography II
Reverse Phase Chromatography
Nonpolar Stationary Phase
Polar Mobile Phase
Column Selection
Mobile-Phase Selection
NO2
Adsorption Chromatography
Classic
Solvent Selection
Non-polar Isomeric Mixtures
Advantages/ Disadvantages
Applications
Ion-Exchange Packings
Types of packings
pellicular bead packing
large (30-40 m) nonporous, spherical, glass,
polymer bead
coated with synthetic ion-exchange resin
sample capacity of these particles is less
coating porous microparticles of silica with a thin film
of the exchanger
faster diffusion leads to enhanced efficiency
Ion-Exchange Equilibria
Exchange equilibria between ions in solution and ions on
CM Cellulose
Cation Exchanger
DEAE Cellulose
Anion Exchanger
ions.
Introduction of a eluent suppressor column
immediately following the ion-exchange column.
Suppressor column
packed with a second ion-exchange resin
Cation analysis
Anion analysis
Size Exclusion
Chromatography(SEC)
Gel permeation(GPC), gel filtration(GFC)
chromatography
Technique applicable to separation of high-molecular
weight species
Rapid determination of the molecular weight or
molecular-weight distribution of larger polymers or
natural products
Solute and solvent molecules can diffuse into pores -trapped and removed from the flow of the mobile phase
SEC(continued)
Specific pore sizes.average residence time in the pores
Disadvantages
Only limited number of bands can be accommodated
Instrumentation
Instruments required:
Schematic of liquid
chromatograph
Elution methods
Isocratic elution
single solvent of constant composition
Gradient elution
2 or more solvents of differing polarity used
Pumping System I
Provide a continuous constant flow of the
Pumping System II
Two types:
constant-pressure
constant-flow
Reciprocating pumps
motor-driven piston
disadvantage: pulsed flow creates noise
advantages: small internal volume (35-400 L), high
output pressures (up to 10,000 psi), ready adaptability
to gradient elution, constant flow rates
LC column
LC injector
tubing
Hundreds of packed columns differing in size and
packing are available from manufacturers ($200$500)
Add columns together to increase length
Liquid Chromatographic
Columns II
Column thermostats
maintaining column temperatures constant to a few
tenths degree centigrade
column heaters control column temperatures (from
ambient to 150oC)
columns fitted with water jackets fed from a constant
temperature bath
Detector
Mostly optical
Equipped with a flow cell
Focus light beam at the center for
analytes
Non-destructive
Types of Detector
Refractive index
UV/Visible
Fluorescence
Conductivity
Evaporative light scattering
Electrochemical
Refractive Index I
Measure displacement of beam with respect to
Refractive Index II
Advantages
universal respond to nearly all solutes
reliable
unaffected by flow rate
low sensitive to dirt and air bubbles in the flow cell
UV/Visible I
Mercury lamp
= 254nm
= 250, 313, 334 and 365nm with filters
Photocell measures absorbance
Modern UV detector has filter wheels for rapidly
UV/Visible II
UV/Visible III
Advantages
high sensitivity
small sample volume required
linearity over wide concentration ranges
can be used with gradient elution
UV/Visible IV
Disadvantage
does not work with compounds that do not absorb light
at this wavelength region
Fluorescence I
For compounds having natural
fluorescing capability
Fluorescence observed by
photoelectric detector
Mercury or Xenon source with
grating monochromator to isolate
fluorescent radiation
Fluorescence II
Advantages
extremely high sensitivity
high selectivity
Disadvantage
may not yield linear response over wide range of
concentrations
Conductivity
Measure conductivity of column
effluent
Sample indicated by change in
conductivity
Best in ion-exchange
chromatography
Cell instability
Electrochemical I
Based on reduction or
Electrochemical II
Advantages
high sensitivity
ease of use
Disadvantages
mobile phase must be made conductive
mobile phase must be purified from oxygen, metal
contamination, halides
Data System
For better accuracy and precision
Routine analysis
pre-programmed computing integrator
Data station/computer needed for higher control levels
add automation options
complex data becomes more feasible
software safeguard prevents misuse of data system
Electrophoresischarged species
migrate in electric field
Separation based on charge or
mobility
Capillary electrophoresis
higher voltages can be used as
the heat can be dissipated
Capillary electrophoresis