analytical techniques 4
TRANSCRIPT
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HPLC
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Outline
What is HPLC? An Overview
Types of HPLC Ion Chromatography Size-Exclusion Chromatography
Adsorption Chromatography
Etc.,
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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 throughthe column
Popularity: sensitivity
ready adaptability to accurate quantitativedetermination
suitability for separating nonvolatile species orthermally fragile ones
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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 identificationof amino acids, proteins, nucleic acids,hydrocarbons, carbohydrates, pesticides,pigments, antibiotics, steroids, and a variety ofother inorganic substances
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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! HPLC - Decrease particle size of packing causes increase in
column efficiency!
diameters 3-10 m
This technology required sophisticated instruments
new method called HPLC
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Instruments required: Mobile phase reservoir
Pump
Injector Column
Detector
Data system
Components
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Elution methods
Isocratic elution single solvent of constant composition
Gradient elution 2 or more solvents
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Provide a continuous constant flow of the solventthrough 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 0.5% orbetter
Pumping System I
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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 flowrates
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Displacement pumps(pulse free 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 bechanged
Flow control and programming system
computer-controlled devices measure flow rate
increase/decrease speed of pump motor
Pumping System III
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Several column types(can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange
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Reverse phase
In this column the packing material isrelatively nonpolar and the solvent is polar
with respect to the sample. Retention is theresult of the interaction of the nonpolarcomponents of the solutes and the nonpolarstationary phase. Typical stationary phases
are nonpolar hydrocarbons, waxy liquids, orbonded hydrocarbons (such as C18, C8, etc.)and the solvents are polar aqueous-organicmixtures such as methanol-water or
acetonitrile-water.
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Size exclusion
In size exclusion the HPLC column isconsisted of substances which have
controlled pore sizes and is able to befiltered in an ordinarily phase according toits molecular size. Small moleculespenetrate into the pores within the packing
while larger molecules only partiallypenetrate the pores. The large moleculeselute before the smaller molecules.
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Ion exchange
In this column type the sample componentsare separated based upon attractive ionicforces between molecules carrying chargedgroups of opposite charge to those chargeson the stationary phase. Separations aremade between a polar mobile liquid, usuallywater containing salts or small amounts of
alcohols, and a stationary phase containingeither acidic or basic fixed sites.
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For injecting the solvent through the column Minimize possible flow disturbances
Limiting factor in precision of liquid chromatographicmeasurement
Volumes must be small
0.1-500 L Sampling loops
interchangeable loops (5-500 L at pressures up to 7000psi)
Sample Injection Systems
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Sample Injection System
The valve handle as shown on the left, the loop is filled from the syringe,
and the mobile phase flows from the pump to column,Valve is placed in position on the right, the loop is inserted
between the pump and the column so that the mobile phase
sweeps the sample onto the column
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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 Column
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Column thermostats maintaining column
temperatures constant to afew tenths degreecentigrade
column heaters controlcolumn temperatures (fromambient to 150oC)
columns fitted with waterjackets fed from a constant
temperature bath
Liquid Chromatographic Columns II
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Mostly optical Equipped with a flow cell
Focus light beam at the center formaximum energy transmission
Cell ensures that the separated
bands do not widen UV-Vis
PDA
Detector
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Some Properties of Detector
Adequate sensitivity
Stability and reproducibility
Wide linear dynamic range
Short response time Minimum volume for reducing zone broadening
High reliability and ease of use
Similarity in response toward all analytes
Selective response toward one or more classes of analytes
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Refractive index
UV/Visible
Fluorescence Conductivity
Evaporative light scattering
Electrochemical
Types of Detector
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Measure displacement of beam with respect tophotosensitive surface of dectector
Refractive Index I
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Advantages
universal respond to nearly all solutes
reliable
unaffected by flow rate
low sensitive to dirt and air bubbles in the flowcell
Refractive Index II
Disadvantages
expensive
highly temperature sensitive
moderate sensitivity
cannot be used with gradient elution
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Mercury lamp
= 254nm
= 250, 313, 334 and 365nm with filters Photocell measures absorbance
Modern UV detector has filter wheels for rapidlyswitching filters; used for repetitive andquantitative analysis
UV/Visible I
Advantages
high sensitivity
small sample volume required
linearity over wide concentration ranges can be used with gradient elution
Disadvantage
does not work with compounds that do not absorb light atthis wavelength region
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For compounds having naturalfluorescing capability
Fluorescence observed byphotoelectric detector
Mercury or Xenon source withgrating monochromator toisolate fluorescent radiation
Fluorescence I
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Advantages
extremely high sensitivity
high selectivity
Disadvantage
may not yield linear response over wide range ofconcentrations
Fluorescence II
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Measure conductivity ofcolumn effluent
Sample indicated bychange in conductivity
Best in ion-exchangechromatography
Cell instability
Conductivity
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Nebulizer converts eluent into mist
Evaporation of mobile phase leads toformation of fine analyte particles
Particles passed through laser beam;
scattered radiation detected at right anglesby silicon photodiode
Similar response for all nonvolatile solutes
Good sensitivity
Evaporative Light Scattering I
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Evaporative Light Scattering II
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Based on reductionor oxidation of the
eluting compoundat a suitableelectrode andmeasurement of
resulting current
Electrochemical I
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Advantages high sensitivity
ease of use
Disadvantages mobile phase must be made conductive
mobile phase must be purified from oxygen,metal contamination, halides
Electrochemical II
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Advantages to HPLC
Quantification Separation Higher resolution and speed of analysis HPLC columns can be reused without
repacking Greater reproducibility due to close control
of the parameters affecting the efficiency ofseparation
Easy automation of instrument operation anddata analysis Adaptability to large-scale, preparative
procedures
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