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Investigation of Ethyl Bridged Hybrid Organic/Inorganic Particles for

Reversed-Phase HPLC Applications

Kevin Wyndham, John O'Gara, Kenneth Glose, Nicole Arpin, Bonnie Alden, Cheryl Boissel,

Pamela Iraneta, and Thomas Walter

HPLC 2002 Conference Montreal Canada

Poster No. 383 June 5, 2002

Waters Corporation

WA20266

KDW 2 © 2002 Waters CorporationWaters

Investigation of Ethyl Bridged Hybrid Organic/Inorganic Particles for Reversed-Phase HPLC Applications

HPLC 2002 Conference Montreal Canada

Poster No. 383 June 5, 2002

We have recently shown that great improvements in chromatographic stability toward high pH mobile phases can be achieved by using hybrid organic/inorganic packing materials. In this report, we take a close look at the synthesis and characterization of highly spherical ethyl bridged hybrids particles. We will examine the effects of using different molar concentrations of ethyl bridged hybrid alkoxysilanes has on the pore properties of these hybrid materials. We will also evaluate the reversed-phase chromatographic performance of these ethyl bridged hybrid packing materials, and compare these results with conventional silica-based packing materials.

Summary

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Hybrid Materials for RP HPLC

Low efficiency

Mechanically weak

Wide pH range

No tailing for bases

Polymeric (Organic)

Tailing peaks for bases

Limited pH range

Mechanically strong

High efficiency

Silica (Inorganic)

UndesirableDesirable

Hybrid Materials seek to combine the desirable attributes of silicaand polymeric materials, and limit the undesirable attributes

Neue, U. D.; et. al. Am. Lab., 1999, 31 (22), 36.Cheng, Y.-F.; et. al. LCGC, 2000, 18 (11), 1162.

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Structure of Ethyl Bridged Hybrids

OSi

OSi

OSi

OSi

OSi

OSi

OSi

OSi

OSi

O

OH OH OH OH OH H2C CH2OH OH

O O O O O O O

OSi O Si

OSi

OSi

OSi

OSi

OSi

OSi

OSi

OH2C CH2 O O O O O O O

Si

SiO

O

O

OH

O

CH2H2C

H2CCH2

Wall

Surface

Increased mechanical stability of columnInternal bridging groups provide high interconnectivity

Increased base stability of columnInternal bridging groups provide hydrophobicity

Reduced USP peak tailing factorsSurface ethyl groups reduce surface silanol concentration

RP HPLC ConsequenceBridged Hybrid Attribute

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Synthesis of Bridged Hybrid Particles

! Explored different ratios inorganic and organic components and surface areas

! Can fully characterize Bridged Hybrids by %C, SEM, TGA, BET, NMR

! Other bridged silanes have been explored

Porous Hybrid Particles

CH2

SiEtOO

SiEtOOEtO

Si

Si

O

OO

O

O

nPolyethoxyoligosiloxane Polymer

CH2EtO

OH

CH2CH2

OEtSi

EtO OEtOEt

CH2SiEtO

OEtEtO

+

Tetraethoxysilane(Inorganic)

Bis(triethoxysilyl)ethane(Organic)

CH2

SiOEt

OEtOEt

Patent Pending

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Influence of Organic Content on Morphology

33 mol% Organic Component 20 mol% Organic Component

! Under standard conditions highly spherical particles were obtained for materials with ≤20 mol% organic component.

! Modified synthetic conditions must be used to prepare spherical particles with >30 mol% organic component.

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Influence of Organic Content on Particle Properties

1850.761455.6%20 mol%

1830.731503.9%11 mol%

2220.721252.4%8 mol%

2710.71971.1%4 mol%

Pore Diameter

(Å)

Pore Volume (cc/g)

Surface Area

(m2/g)%C

Organic Content

Notes: %C determined by elemental analysis. Surface propertieswere determined by Nitrogen Sorption (BET Method).

! Higher organic content in Bridged Hybrids leads to greater carbon content and surface areas.

! Since 20 mol% is the highest organic content with desirable morphology, we explored this material in depth.

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Different Surface Areas of 20 mol% Organic Content Bridged Hybrids

Pore Diameter

Pore Volume

Surface Area 189 m2/g171 m2/g145 m2/g

148 Å150 Å185 Å

0.76 cc/g0.71 cc/g0.72 cc/g

BridgedHybrid C

BridgedHybrid B

Bridged Hybrid A

5 µm Bridged Hybrid

! We can prepare a variety of 20 mol% organic content Bridged Hybrids (sized 5 µm) with different surface areas, from modifications in the synthetic procedure.

! These Bridged Hybrids are highly spherical, and have comparable characterization data (%C, SEM, TGA, NMR)

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C18-Surface Bonding of 5 µm Particles

3.0 µmol/m2

14.0 %

185 Å

0.72 cc/g

145 m2/g

5.6%

Bridged Hybrid A

3.2 µmol/m2

15.9 %

150 Å

0.71 cc/g

171 m2/g

5.7%

BridgedHybrid B

0.85 cc/g0.76 cc/gPore Volume

3.3 µmol/m23.0 µmol/m2C18 Coverage

19.8 %16.7 %Total %C

100 Å148 ÅPore Diameter

340 m2/g189 m2/gSurface Area

0.0%5.9%Particle %C

SilicaBridgedHybrid C

2. End Cap

1. C18 BondO Si

O

OOH

! Slightly lower surface coverages are expected for Bridged Hybrids due to reduced surface silanol concentrations

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Chromatographic Characterization

NH

HN O

O

Uracil

Naphthalene Acenaphthene

Butyl Paraben

Propranolol pKa = 9.6Amitriptyline pKa = 9.4

OH

O OC4H9

O

OH

NH2+

NH+

OC3H7

O

OC3H7

ODipropylphthalate

Mobile Phase: Flow Rate: Temperature:

65:35 Methanol / 20 mM KH2PO4 Buffer pH 7.01.4 mL/min, 4.6 x 150 mm23.4± 0.1 ºC

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Comparison of C18-bondings on Bridged Hybrids and Silica

! Increased retentivity with higher surface area Bridged Hybrids! Bridged Hybrid C18 columns have similar selectivity as Silica C18 columns,

but better peak shape for basic analytes

10 20 30

Silica

Time (min)

V0

12 3 4

65 Bridged Hybrid A

Bridged Hybrid B

Bridged Hybrid C

V0. Uracil1. Propranolol2. Butylparaben3. Dipropylphthalate4. Naphthalene5. Acenaphthene6. Amitriptyline

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Accelerated High pH Stability Tests

TEST A: pH 10 (Performed at 50 °C)

" Challenge: 50 mM NEt3, pH 10, 2 mL/min for 60 min# Wash Step: water (2 mL/min ) for 10 min, methanol (2 mL/min) for 10 min$ Test: Methanol / pH 7.0 K2HPO4 (65/35 v/v)% Return to 1 until column fails

Column failure:

TEST B: pH 12 (Performed at 50 °C)

" Challenge: 0.01 M NaOH, pH 12, 2 mL/min for 60 min# Wash Step: water (2 mL/min ) for 10 min, methanol (2 mL/min) for 10 min$ Test: 50% acetonitrile in water% Return to 1 until column fails

Defined as time (hrs) until >50% loss in plate number (N), or when column voided

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High pH Stability Test A (pH10): Comparison of Bonded Silica and Bridged Hybrids

2030405060708090

100110

0 25 50 75 100 125 150

Time Exposed to pH 10 Buffer (hrs)

%O

rigin

al E

ffic

ienc

y (5

sig

ma)

B ridged Unbo ndedB ridged C 18Silica C 18 - B rand ASilica C 18 - B rand B

! C18-bonded and unbonded 20 mol% organic content Bridged Hybrid columns have an order of magnitude greater base stability than Silica C18

! Where Silica columns fail from rapid dissolution of SiO2, bonded phases on Bridge Hybrids may hydrolyze before dissolution of the particle

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High pH Stability Test B (pH 12): Surface Area Contribution to Stability of Bridged Hybrids

R2 = 0.9274

R2 = 0.8668

0

20

40

60

80

100

120

140 150 160 170 180 190 200

Surface Area (m2/g)

Effi

cien

cy h

alf

life

pH 1

2 bu

ffer

(ho

urs)

Bridged C18

Bridged Unbonded

! Silica C18 columns fail within an hour on this test! C18-bonded and unbonded 20 mol% organic content Bridge Hybrid

columns last > 20h! General trends for Bridged Hybrid C18 columns:

↓ Surface Area ↑ Base Stability

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Summary

! A variety of Bridged Hybrid material, with different surface areas and organic content can now be prepared

! Chromatographic Properties of 20 mol% organic content Bridged Hybrids C18 columns were assessed:! Similar selectivity as Silica C18 columns! Reduced peak tailing for basic analytes

! At least an order of magnitude higher column stability in high pH mobile phases

! Column stability toward high pH increases with decreasing surface area

Contact InformationKevin Wyndham

kwyndham@waters.com