types of liquid chromatography
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Types of Liquid ChromatographyTypes of Liquid Chromatography
I. Ion Exchange ChromatographyA. Factors influencing retentionB. Suppressed ion exchange
II. Partitioning ChromatographyA. Normal phase/ reverse phase
III. Size Exclusion ChromatographyIV. Supercritical Fluid Chromatography/ SFEV. Capillary Electrophoresis
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Factors Influencing retention in Ion Factors Influencing retention in Ion ExchangeExchange
• Ionic strength: not real important in selectivity
• pH: anion exchange pH retention cation exchange pH retention
• Temperature: T efficiency Flow rate: Slightly slower than other HPLC methods to maximize resolution & improve mass transfer kinetics
• Buffer salt: Influences pH & selectivity
• Organic Modifier: Solvent strength increases with increases in modifier
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Suppressed Ion ChromatographySuppressed Ion Chromatography
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Partitioning ChromatographyPartitioning Chromatography
• Analyte interacts with mobile and stationary phase, differential interaction leads to selectivity
• Interactions that are important– Proton accepting ability * most important– Dipole interaction– Proton Donor * most important– e- pair donating ability– Van der Waals dispersion forces
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Types of Partitioning Types of Partitioning ChromatographyChromatography
Normal PhaseStationary phase: Polar
with short carbon chains
Mobile phase: Non-polar such as hexane
Polar things are retained on column
Applications: oil soluble vitamins, nitrophenols
Example Stationary Phase
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Types of Partitioning Types of Partitioning ChromatographyChromatography
REVERSE PHASEMore common
Stationary Phase: Hydrophobic C18 or C8
Mobile Phase:
Polar usually aqueous
Polar substance elute first
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Solvophobic TheorySolvophobic Theory
• Water has a lot of intermolecular interactions in the liquid phase
• Solute dissolved in water disrupts those intermolecular interactions
• Solute is forced out of aqueous phase not because of favorable interactions between analyte and stationary phase but because of unfavorable interactions between solute and water when solute is dissolved in aqueous phase hence: SOLVOPHOBIC THEORY
• Polar functional groups such as –OH would increase the favorability of interaction and thus decrease retention (in mobile phase longer)
• Polar things elute non-polar things elute
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Sample Column Packing
Mobile phase
C-18
C-8
C-2
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Size Exclusion ChromatographySize Exclusion Chromatography
• Molecules partition into bead• Large molecules can’t get in and
are unretained, small molecules get in and never get out, medium size will differentiate
• Need at least 10% difference in MW to differentiate
• GPC organics• Gel filtration chromatography
aqueous
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Size Exclusion ChromatographySize Exclusion Chromatography
Advantages
1) Short, well defined retention times
2) Narrow bands high sensitivity
3) No sample loss b/c no interaction with stat. phase
4) No column destruction b/c no interaction with stat. phase
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Size Exclusion ChromatographySize Exclusion Chromatography
Disadvantages1) Only limited # of
peaks can be separated b/c time scale of separation is short
2) Not good for separating compounds of similar size
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SummarySummary
Phase/ Mode % Use
Reverse phase 50.6
Normal phase 24.1
Ion Exchange 14.1
Size Exclusion 6.6
Chiral 3.5
Hydrophobic 1.1
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Supercritical FluidsSupercritical Fluids
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Supercritical Fluid Chromatography Supercritical Fluid Chromatography InstrumentationInstrumentation
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Properties of Mobile Phases Used Properties of Mobile Phases Used in Chromatographyin Chromatography
Mobile Phase
Density (g/mL) Viscosity (poise 10-4)
Diffusion coefficient (cm2/sec)
Gas 0.6 – 2.0 x 10-3 0.5 – 3.5 0.01 – 1.0
Super-critical fluid
0.2 – 0.9 2.0 – 9.9 0.5 – 3.3 x 10-4
liquid 0.8 – 1.0 30 -240 0.5 – 2.0 x 10-5
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Fluid Dipole moment
Tc(oC) Pc(atm) Densityc(g/mL)
Density400(g/mL)
CO2 0 31.3 72.9 0.47 0.96
N2O 0.51 36.5 72.5 0.45 0.94
NH3 1.65 132.5 112.5 0.24 0.40
N-C5 0 196.6 33.3 0.23 0.51
N-C4 0 152.0 37.5 0.23 0.50
SF6 0 45.5 37.1 0.74 1.61
Xe 0 16.6 58.4 1.10 2.30
CCl2F2 0.17 111.8 40.7 0.56 1.12
CHF3 1.47 25.9 46.9 0.52
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Supercritical fluid extraction (SFE)Supercritical fluid extraction (SFE)
Used instead of soxhlet extraction
Advantages
1. Fast: rate of diffusion between sample matrix & extraction fluid 10-60 min vs. days
2. Solvent strength can be varied by changes in P & T
3. Less Harmful solvent
4. Many SCF are gases at RT, recovery of analytes is easy
5. Many SCF are cheap, inert, and non-toxic
6. On-line extraction
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Supercritical fluid extractionSupercritical fluid extraction
• Disadvantages1. Method development
is more complex2. Limited # of mobile
phases3. Capital equipment &
CO2 expensive4. Requires more
operator time to do 1 at time
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• Insert Hawthorne paper