1 hplc columns and stationary phases lecture 2 yuri kazakevich seton hall university
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HPLC Columns andStationary Phases
Lecture 2
Yuri KazakevichSeton Hall University
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Packing material• Particle type• Particle geometry• Surface chemistry
Bonded Layer• Chemistry• Conformational freedom• Interaction with solvent
Outline
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Silica
rigid porous (or nonporous) particles
wide variety of particle and pore sizes
soluble in water at pH > 8
from : Journal of Chromatography A, 1006 (2003) 207–228
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HPLC Adsorbent Particles
• Average particle diameter is 5 • Average pore diameter is 100 Å• Average surface area is 300 m2/g
DLS
Most of the adsorbents have cylindrical pore shape.What is the ratio of particle diameter to the pore diameter?
What is the total length of all pores in 1 g of adsorbent?
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Packing MaterialPore size, pore volume, surface area
• Assuming cylindrical pore model one can get:
• The larger the pore diameter, the smaller the surface area.• The larger the surface area the greater the retention.• The smaller the pore diameter the greater the steric hindrance effect.
V
S
D
4DLSL
DV
4
2
Analyte retention in HPLC is proportional tothe surface area of packing material
SKVVR 0
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Bonded Phase
• Bonded phase shields polar silica surface, making it inaccessible for analyte molecules.
• Suppressing strong polar interactions with silica surface and substituting them with weak dispersive forces is a key factor of reversed-phase HPLC.
• BP types - C18, C8, C5, C1, Phenyl, CN, NH2,
etc.
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Selected Types of Bonded Ligands
C-18 C8
C1
PFPOxy-Phenyl
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C18 Ligands
C18 chains have ~21 Å length in all-trans conformation
Their molecular volume is ~700 Å3
Maximum bonding densityis 2.5 chains/nm2 or 4.1 mole/m2 on flat surface
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Allure-C18
Allure-PFP
Zorbax-C8
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
3
-2 0 2 4 6 8
number of carbon atoms
ln(k
')
Alkylbenzenes from MeCN/Water (80/20)
Methylene selectivity vs. eluent composition
0
0.1
0.2
0.3
0.4
0.5
0.6
50 60 70 80 90 100
MeCN/Water %v/v
Slo
pe
ln (
k')
Zorbax-C8
Allure-C18
Allure-PFP
Methylene Selectivity of Different Bonded Phases
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Monomeric and Polymeric Bonding
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Endcapping
Secondary bonding with trimethylchlorosilane
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Propyl-Phenyl ligands at 2.7 group/nm2 (left) and 1.9 group/nm2 (right)
Bonding Density
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Bonding Density
• The only measurable parameter related to the quality of bonded phase is Carbon Loading (%w/w of carbon atoms bonded on the silica surface).
• Bonding density is the number of bonded ligands per unit of silica surface.
Carbon Load(%w/w)
Bonding Density(uMole/m2)
Nonporoussilica, S=4 m2/g
0.24 4.0
Porous silica,S=300 m2/g
12 2.3
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Arrangement of the Bonded Phase Chains on Silica Surface
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S
V LayerBond . S
RVRR LB ..2 2For flat surface
For concavesurface
A B
Bonded Layer Thickness
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Column Pore Volume
0.7
0.9
1.1
1.3
1.5
1.7
1.9
0 5 10 15
Carbon number
Vo
lum
e [
ml/
colu
mn
]
ColumnPore Volume
InterparticleVolume
.0 exclpore VVV
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0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15
carbon number, nVo
lum
e [u
l/m2]
Free volume
MeCN ads. Vol.
MeOH ads. Vol.
THF ads. Vol
0
2
4
6
8
10
12
14
16
0 5 10 15 20
Carbon number
Ad
s.
La
ye
r T
hic
kn
es
s [
Å]
MeCN
MeOH
THF
]/[]/[ .max moleLVmmolen molads 2
Volume and Thickness of Adsorbed Layer on All Studied Adsorbents
for Three Adsorbates
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-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
3
20 30 40 50 60 70 80 90 100
MeOH/Water MeCN/Water
-4
-3
-2
-1
0
1
2
3
4
0 20 40 60 80 100
MeOH [v/v%]
Eluent Type Effect
MeCN [v/v%]
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Retention Model
HPLC analyte injected in the column equilibrated with binary eluent
Assumption: Small amount of analyte does not significantly disturb eluent equilibrium in the column
Overall retention is acomposition of twoconcurrent processes
Column PerformancepH stability
• The main parameter affecting pH stability of packing material is Bonding Density
• Low pH (<2.5) causes hydrolysis of the siloxane bonds destroying bonded layer– The higher the bonding density the lower hydrolysis effect.
• High pH (>8.5) causes silica dissolution– High bonding density shield silica surface which makes it
stable up to pH 13.
Column Testing
• Good reversed-phase column should – exclude acidic components (benzoic acid should come
out before Vo),
– show low retention and tailing for basic components (pyridine)
– show complete separation and very symmetrical peaks for naphthalene and ethylbenzene.
• Testing conditions: Acetonitrile/water 70/30, 1 ml/min.
Column Testing
Prodigy-ODS SupelcoSil-LC18
Ben
zoic
aci
d Ben
zoic
aci
d
Pyri
dine
Pyri
dine
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H O2 MeCN H O2 MeCN H O2 MeCN H O2 MeCN H O2 MeCN H O2 MeCNH O2 MeCN
Column Cleaning
Solvent front disturbs phase equilibrium Release of trapped impurities
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Surface area• Analyte retention is proportional to the adsorbent surface
areaPore size
• Effects the conformational freedom of bonded ligands• Restricts the pore volume accessibility for large molecules• Minor effect on the amount of accessible residual silanoles
Type of bonded ligands• Determines the adsorbent retentive power and selectivity
Bonding density• Determines the accessibility of residual silanoles• Minor effect on the selectivity
Summary