INSTRUMENTAL ANALYSIS CHEM 4811 CHAPTER 13 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of chemistry Department of natural sciences Clayton state university

LIQUID CHROMATOGRAPHY (LC) 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

ELUENT STRENGTH (εo) - Weakly retained solutes are first eluted with a low 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 contribution HCs < ethers < esters < ketones < aldehydes < amides < amines < alcohols

LIQUID CHROMATOGRAPHY (HPLC) 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 phase Amino [–(CH2)3NH2] Cyano [–(CH2)3–C≡N] Diol [–(CH2)2OCH2CH(OH)CH2OH]

REVERSE PHASE HPLC (RP-HPLC) - 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 phase Octadecyl [–(CH2)17CH3; C18,ODS] Octyl [–(CH2)7CH3; C8] Phenyl [–(CH2)3C6H5]

HPLC INSTRUMENTATION - 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 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 surface of a solid support - Mobile phase is a liquid or a gas - Solute equilibrates between the stationary and mobile phases

SUPERCRITICAL FLUID CHROMATOGRAPHY (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

CHIRAL CHROMATOGRAPHY - 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

ELECTROPHORESIS - Migration of ions in an electric field - Cations are attracted to the negative electrode (cathode) - Anions are attracted to the positive electrode (anode)

Capillary Electrophoresis (CE) 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

CE BACKGROUND ELECTROLYTE (RUN BUFFER) - A buffer solution in the electrode reservoirs - Controls pH and ionic strength

PRINCIPLES OF CE - 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 nL of 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

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

MODES 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)

DETECTORS Similar to those of HPLC - Ultraviolet absorption (most common) - Conductivity - Electrochemical - Fluorescence