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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