Transcript High Performance Liquid Chromatography

Gas Chromatography
Gas Chromatography
 an analytical separations technique useful
for separating volatile organic compounds
 consists of :
– Flowing mobile phase (inert gas - Ar, Ne, N)
– Injection port ( rubber septum - syringe injects
sample)
• kept at a higher temperature than the boiling point
Principles
 Separation due to differences in partitioning
behavior
 selective retardation
Key Information
 organic compounds separated due to
differences in their participating behavior
between the mobile gas phase and the
stationary phase in the column
 in contrast to other types of
chromatography, the mobile phase does not
interact with molecules of the analyte; its
only function is to transport the analyte
through the column
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
Martin and Synge (also did liquid partition
chromatography)
 10+ years later GC used experimentally
 1955, first commercial apparatus for GC
appeared on the market
Today
 estimate : 200, 000 gas chromatographs are
currently used through out the world.
 30+ instrument manufactures
 130 different models
 cost 1,500 to 40,000 dollars
 improvements: computers- automatic
control open tubular columns-separate a
multitude of analytes in relatively short
times
Uses of Gas Chromatography
 Determination of volatile compounds (gases
& 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
Liquid liquid extraction
repeated extraction is basis
for LC
Retardation of solutes in liquid
onto a solid phase
Elution chromatography
 Increasing polarity
Solvents mixed
of pure solvents
 hexane
 ether
 acetone
 methanol
 water
 acetic acid
%hexane and %
methanol
miscible
can be mixed
continuously
(solvent
programming)
Types of Liquid Chromatography
 Liquid-solid: adsorption on solid
which is generally polar (silica gel,
alumina, magnesium silicates) or
reverse phase (cellulose, poly
amides)
Ion exchange: specific interactions
with ionic species (change relative
strengths of acid or base)
Types of Liquid Chromatography
Liquid-liquid: partition between 2
bulk phases (one immobilized) is
highly selective
Liquid exclusion: molecular sieve
separates molecules on basis of
ability to diffuse into immobile
support
Retardation based on size of
molecule as it diffuses into
porous solid
High Performance Liquid
Chromatography
Once called High Pressure Liquid
Chromatography
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
amino acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, pharmaceuticals, pesticides, pigments,
antibiotics, steroids, and a variety of other inorganic
substances
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:
Si
O
Si
O
O
OH
O
OH
O
Si
CH3
Si
R
CH3
Si
R= C8, C18
Partition Chromatography II
 Reverse Phase Chromatography
– Nonpolar Stationary Phase
– Polar Mobile Phase
 Normal Phase Chromatography
– Polar Stationary Phase
– Nonpolar Mobile Phase
 Column Selection
 Mobile-Phase Selection
Partition Chromatography III
 Research Applications
– Parathion in Insecticides:
O
– CH3CH2O P O
CH3CH2O
NO2
– Cocaine in Fruit Flies: A Study of
Neurotransmission by Prof. Jay Hirsh, UVa
Adsorption Chromatography
 Classic
 Solvent Selection
 Non-polar Isomeric Mixtures
 Advantages/ Disadvantages
 Applications
What is Ion Chromatography?
 Modern methods of separating and determining ions
based on ion-exchange resins
 Mid 1970s
 Anion or cation mixtures readily resolved on HPLC
column
 Applied to a variety of organic & biochemical systems
including drugs, their metabolites, serums, food
preservatives, vitamin mixtures, sugars,
pharmaceutical preparations
The Mobile Phases are...
 Aqueous solutions
– containing methanol, water-miscible organic solvents
– also contain ionic species, in the form of a buffer
– solvent strength & selectivity are determined by kind
and concentration of added ingredients
– ions in this phase compete with analyte ions for the
active site in the packing
Properties of the Mobile Phase
 Must
– dissolve the sample
– have a strong solvent strength leads to reasonable
retention times
– interact with solutes in such a way as to lead to
selectivity
Ion-Exchange Packings
 Types of packings
– pellicular bead packing
• large (30-40 µm) 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
the surface of an insoluble, high molecular-weight solid
 Cation exchange resins
– sulfonic acid group, carboxylic acid group
 Anion exchange resins
– quaternary amine group, primary amine group
CM Cellulose
Cation Exchanger
DEAE Cellulose
Anion Exchanger
Eluent Suppressor Technique
 Made possible the conductometric detection of eluted
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
depends on the effective size of the analyte molecules
– larger molecules
– smaller molecules
– intermediate size molecules
SEC Column Packing
 Small (~10 µm) silica or polymer particles containing
a network of uniform pores
 Two types (diameters of 5 ~ 10 µm)
– Polymer beads
– silica-based particles
Advantages of Size Exclusion
Chromatography
 Short & well-defined separation times
 Narrow bands--> good sensitivity
 Freedom from sample loss, solutes do not interact
with the stationary phase
 Absence of column deactivation brought about by
interaction of solute with the packing
Disadvantages
 Only limited number of bands can be accommodated
because the time scale of the chromatogram is short
 Inapplicability to samples of similar size, such as
isomers.
– At least 10% difference in molecular weight is required
for reasonable resolution
Instrumentation
 Instruments required:
–
–
–
–
–
–
Mobile phase reservoir
Pump
Injector
Column
Detector
Data system
Schematic of liquid
chromatograph
Mobile phase reservoir
 Glass/stainless steel reservoir
 Removal of dissolved gases by degassers
– vacuum pumping system
– heating/stirring of solvents
– sparging
– vacuum filtration
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
solvent through the injector
 Requirements
–
–
–
–
pressure outputs up to 6000 psi
pulse-free output
flow rates ranging from .1-10 mL/min
flow control and flow reproducibility of
.5% or better
– corrosion-resistant components
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
Pumping System III
 Displacement pumps
– syringe-like chambers activated by screw-driven
mechanism powered by a stepper motor
– advantages: output is pulse free
– disadvantage: limited solvent capacity (~20 mL) and
inconvenience when solvents need to be changed
 Flow control and programming system
– computer-controlled devices
– measure flow rate
– increase/decrease speed of pump motor
Sample Injection Systems
 For injecting the solvent through the column
 Minimize possible flow disturbances
 Limiting factor in precision of liquid chromatographic
measurement
 Volumes must be small
 .1-500 L
 Sampling loops
– interchangeable loops (5-500 L at pressures up to
7000 psi)
LC column
LC injector
Liquid Chromatographic Column
 Smooth-bore stainless steel or heavy-walled glass
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
maximum energy transmission
 Cell ensures that the separated
bands do not widen
Some Properties of Detector
 Adequate sensitivity
 Stability and reproducibility
 Wide linear dynamic range
 Short response time
 Minimum volume for reducing zone broadening
More Properties of Detector
 High reliability and ease of use
 Similarity in response toward all analytes
 Selective response toward one or more classes of
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
photosensitive surface of dectector
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
Refractive Index III
 Disadvantages
– expensive
– highly temperature sensitive
– moderate sensitivity
– cannot be used with gradient elution
UV/Visible I
 Mercury lamp
  = 254nm
  = 250, 313, 334 and 365nm with filters
 Photocell measures absorbance
 Modern UV detector has filter wheels for rapidly
switching filters; used for repetitive and quantitative
analysis
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
Evaporative Light Scattering I
 Nebulizer converts eluent into mist
 Evaporation of mobile phase leads to formation of fine
analyte particles
 Particles passed through laser beam; scattered
radiation detected at right angles by silicon
photodiode
 Similar response for all nonvolatile solutes
 Good sensitivity
Evaporative Light Scattering II
Electrochemical I
 Based on reduction or
oxidation of the eluting
compound at a suitable
electrode and measurement of
resulting current
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
Electrophoresis…charged 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