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

2. LIQUID CHROMATOGRAPHY (LC)
CHAPTER 13
LIQUID CHROMATOGRAPHY (LC)

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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

9. 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]

10. 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]

11. 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)

12. 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

13. 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

14. 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

15. 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)

16. HPLC HYPHENATED TECHNIQUES
HPLC-UV-VIS
LC-NMR
HPLC-MS (THERMOSPRAY INTERFACE)
HPLC-MS (ELECTROSPRAY INTERFACE)
HPLC-MS (APCI INTERFACE)

17. 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

18. 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

19. 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

20. ADSORPTION CHROMATOGRAPHY
- Stationary phase is a solid
- Mobile phase is a liquid or a gas
- Solute adsorbs to the surface of the solid particles

21. 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

22. 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

23. 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

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

25. 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

26. 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

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

28. 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

29. 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)

30. 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

31. PRINCIPLES OF CE At High pH
- Electroosmosis is faster than electrophoresis
- Net flow of anions is towards the cathode
At Neutral pH

32. 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

33. 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

34. 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)

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