INSTRUMENTAL ANALYSIS CHEM 4811
CHAPTER 13
DR. AUGUSTINE OFORI AGYEMANAssistant professor of chemistryDepartment of natural sciences
Clayton state university
CHAPTER 13
LIQUID CHROMATOGRAPHY (LC)
LC STATIONARY PHASE
- Usually silica (SiO2 · xH2O)
- Alumina (Al2O3 · xH2O)
- Adsorption of water slowly deactivates adsorption sites of silica
- May be reactivated by heating to about 200oC in an oven
ELUENT STRENGTH (εo)
- A measure of adsorption energy of solvent
- εo of pentane is 0
- More polar solvents have greater eluent strengths
- Solutes elute more rapidly when eluent strength is greater
- Weakly retained solutes are first eluted with alow eluent strength solvent
- Eluent strength is increased by adding a polar solvent to elute strongly retained solutes
- Eluent strength is increased by making mobile phase more like the stationary phase
Increasing order of polarity contributionHCs < ethers < esters < ketones < aldehydes < amides
< amines < alcohols
ELUENT STRENGTH (εo)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
- Good for Proteomics (analysis of amino acids and peptides)
- Uses closed packed columns under high pressure
- Resolution increases with decreasing particle size of stationary phase
- Decreasing particle size decreases plate height
- Small particle size reduces the terms A and C in the van Deemter equation
HPLC STATIONARY PHASE
- Microporous spherical particles of silica (2 – 10 µm)
- Long chain C18 hydrocarbon bonded to silica surface (Octadecyl)
- Particles must be stable, uniform, spherical for uniform flow
- The area covered by stationary phase is greater than that of the polymer film in GC
- Column operates at very high pressures (500 – 6000 psi)
HPLC STATIONARY PHASE
- The organosilyl bonds may hydrolyze at very low pH
- Silica may dissolve at high pH (above 8)
- Silica can become soluble at very high temperatures
- Zirconia (ZrO2) is used as particle support which has greater pH range stability (1 – 14) and temperature stability (> 100 oC)
- Cross-linked styrene-divinylbenzene copolymer (called XAD resins) are also used
NORMAL PHASE HPLC
- Stationary phase is polar
- Mobile phase is less polar
- Eluent strength is increased by adding a more polar solvent
Examples of organic substituents for stationary phaseAmino [–(CH2)3NH2] Cyano [–(CH2)3–C≡N]
Diol [–(CH2)2OCH2CH(OH)CH2OH]
- More common
- Stationary phase is weakly polar or nonpolar
- Mobile is more aqueous or polar
- Insensitive to polar impurities and eliminates tailing
Examples of organic substituents for stationary phaseOctadecyl [–(CH2)17CH3; C18,ODS]
Octyl [–(CH2)7CH3; C8] Phenyl [–(CH2)3C6H5]
REVERSE PHASE HPLC (RP-HPLC)
- Mobile Phase Degassing (reservoirs with He gas for removal of dissolved gases)
- Mobile Phase Storage (reservoirs for storing mobile phase)
- Mobile Phase Mixing (for mixing solvents from reservoirs)
- HPLC Pump (generates high pressure for high performance)
HPLC INSTRUMENTATION
- Fill/Drain Valve (liquid transfer lines and components)
- Rotary Sample Loop Injector (introduces sample with syringe)
- The Column- Irreversible adsorption to the columns is very common
- Disposable guard column containing the same stationary phase is attached to the column entrance
- HPLC Detectors
HPLC INSTRUMENTATION
Isocratic Elution- Elution with a single solvent
- Elution with a constant solvent mixture
Gradient Elution- Solvent is changed continuously from weak to
strong eluent strength
- Used when the solvent does not efficiently elute all components
HPLC INSTRUMENTATION
HPLC DETECTORS
Refractive Index (RI) Detector- Universal, nondestructive, concentration detector
- Relatively insensitive and for only isocratic separations
Evaporative Light Scattering Detector (ELSD)- Universal, destructive, mass-flow detector
- Sensitive and for isocratic or gradient separations
UV-VIS Absorption Detectors (most common)- Nondestructive, concentration detector
- Varying sensitivity and for isocratic or gradient separations
HPLC DETECTORS
Fluorescence Detector- Compound specific, nondestructive, concentration detector- Varying sensitivity and for isocratic or gradient separations
Electrochemical Detectors (ECD)- Compound specific, destructive, concentration or mass-flow
- Varying sensitivity and for isocratic RP-HPLC
Conductometric Detector-For cations and anions in solution, nondestructive, concentration
- For isocratic RP-HPLC and ion chromatography
Charged Aerosol Detector (most sensitive)
HPLC HYPHENATED TECHNIQUES
HPLC-UV-VIS
LC-NMR
HPLC-MS (THERMOSPRAY INTERFACE)
HPLC-MS (ELECTROSPRAY INTERFACE)
HPLC-MS (APCI INTERFACE)
ION-EXCHANGE CHROMATOGRAPHY
- Allows separation of ions and polar molecules
- Ionic groups are covalently attached to a stationary solid phase
- Mobile phase is a liquid
- Ionic solutes are electrostatically attracted to the stationary phase
AFFINITY CHROMATOGRAPHY
- Very selective
Powerful for separating a single biomolecule from a complex mixture of biomolecules
- Based on specific interactions between a type of solute molecule and another molecule covalently
attached to the stationary phase
SIZE EXCLUSION CHROMATOGRAPHY
- Also known as Gel Filtration or Gel Permeation
- Solutes are separated based on size
- Stationary phase has small pores that exclude large molecules
- Small molecules enter the pores so spend more time in column
- Large molecules come out of column before small molecules
ADSORPTION CHROMATOGRAPHY
- Stationary phase is a solid
- Mobile phase is a liquid or a gas
- Solute adsorbs to the surface of the solid particles
PARTITION CHROMATOGRAPHY
- Stationary phase is a thin liquid coated on the surfaceof a solid support
- Mobile phase is a liquid or a gas
- Solute equilibrates between the stationary and mobile phases
SUPERCRITICAL FLUIDCHROMATOGRAPHY (SFC)
- Applies the ability of supercritical fluids to dissolve poorly volatile molecules
- Stationary phase is commonly used column packings
- Mobile phase is a supercritical fluid (CO2, C2H6, N2O)
- For extracting caffeine from coffee beans and nicotine from cigarette tobacco
- Employs chiral phases to separate enantiomers
- Cyclodextrins (complex cyclic carbohydrates) bind enantiomers at different strengths enabling separation
- The cyclodextrin pockets come in different sizes which is suitable for separation of enantiomer pairs of different sizes
- Relative amount of each enantiomer can also be determined
- Chiral GC columns are also available
CHIRAL CHROMATOGRAPHY
ELECTROPHORESIS
- Migration of ions in an electric field
- Cations are attracted to the negative electrode (cathode)
- Anions are attracted to the positive electrode (anode)
ELECTROOSMOSIS
- The application of electric field to drive fluid in a capillary tube from anode towards cathode
Capillary Electrophoresis (CE) - Is a combination of electrophoresis and electroosmosis
CAPILLARY ELECTROPHORESIS(CE)
- High-resolution separation method
- Separates charged and neutral analytes in a narrow capillary tube
- Capillary tube wall is usually fused silica
Good technique for the separation of- Small ions (Na+, K+)
- Proteins- DNA
- A buffer solution in the electrode reservoirs
- Controls pH and ionic strength
CE BACKGROUND ELECTROLYTE (RUN BUFFER)
- Both ends of the capillary tube is first immersed in a background electrolyte
- One end of tube is dipped in vial containing the sample
- Pressure or electric field is applied to introduce ~10 nLof sample into the capillary
- Capillary is placed back into the electrolyte
- 20 to 30 kV is applied and causes ions in capillary to migrate
PRINCIPLES OF CE
- Different ions migrate at different speeds
- This results in separation of ions
- Ions reach a detector and an electropherogram (response versus time) is recorded
- Very narrow bands are usually seen
- Terms A and C in the van Deemter equation are reduced(no multiple paths and no stationary phase)
PRINCIPLES OF CE
- Electroosmosis sweeps analyte molecules towards the detector
- Detector is placed near the cathode
- Detector is set to a wavelength of about 200 nm
- Cations reach the detector first
- Neutral molecules reach the detector after cations
- Anions reach the detector after neutral molecules
PRINCIPLES OF CE
At High pH- Electroosmosis is faster than electrophoresis
- Net flow of anions is towards the cathode
At Neutral pH- Electroosmosis is faster than electrophoresis
- Net flow of anions is towards the cathode
PRINCIPLES OF CE
At Low pH- Electroosmosis is slower than electrophoresis
- Net flow of anions is towards the anode
- Anions may not reach the detector
- Polarity can be reversed to separate anions
PRINCIPLES OF CE
Capillary Ozone Electrophoresis (CZE)- Separation is based on different velocities of different ions
Capillary Gel Electrophoresis (CGE)- Separation is based on size of molecules
- Molecules are separated upon migrating through a gel in the capillary column
- The process is called sieving- Small molecules travel faster than large molecules
MODES OF CE
Cpillary Isoelectric Focusing (CIEF)- Used separate proteins, peptides, amino acids
- Basis is the presence of both the acidic and basic groups- Separated is based on different isoelectric points by varying pH- Compounds exist as zwitterion with no net charge at a given pH
Micellar Electrokinetic Capillary Chromatography (MEKC)- Used to separate different neutral molecules
- Also used for ions- Micelles are added to the background electrolyte
- An example is sodium dodecyl sulfate (a surfactant)
MODES OF CE
Similar to those of HPLC
- Ultraviolet absorption (most common)
- Conductivity
- Electrochemical
- Fluorescence
DETECTORS