Understanding Stationary Phases for Reversed-Phase ... · (1990) 573-582 Accessible Surface Ligand...
Transcript of Understanding Stationary Phases for Reversed-Phase ... · (1990) 573-582 Accessible Surface Ligand...
Patrick D. McDonald*, Bonnie A. Alden,KimVan Tran, Charles H. Phoebe, Jr.,Pamela C. Iraneta, Mark Capparella,Thomas H. Walter, Uwe D. Neue,Barbara K. Grover, John E. O’Gara,
Joseph C. Arsenault, Yuehong Xu, Pamela A. Richards*[email protected]
Waters Corporation, 34 Maple Street, Milford, MA USA 01757Poster: HPLC 2001 Maastricht, 18-19 June 2001
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Understanding Stationary Phasesfor Reversed-Phase Separations:New Notions for a New Century
Dr. Pat McDonald
[1980]Barbara Grover
Joe Arsenault
ChemicalProducts
Analysis
Evaluation
Chem R&D
Dr. Tom Walter
SynthesisDr. John O'Gara
Dr. Yuehong Xu
Pam Richards
Pam Iraneta
Mark Capparella
Bonnie Alden
Dr. Chuck Phoebe Dr. Uwe NeueKimVanTran
Applications
Info Center
Carla ClaytonGrace LavalleeMaureeen Allegrezza
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Thank You Colleagues! 2
Experiments designed to explore the actual physical reality of the interaction of mobile andstationary phases are fraught with practical limitations that impede drawing significantconclusions from the results. It has been easier for most practitioners to foster the folkloreforged through the citation generations of HPLC literature than to refine the "cartoon-level"view of the chromatographic process to a higher "art".
Hybrid Particle Technology enables creation of chromatographic substrates with interesting,specifically designed chemical modifications that reside not simply at the "accessible" surface,but, rather, throughout the entire backbone of a particle’s molecular make-up.This now permits rational exploration of structure-activity relationships with a view towardunderstanding and explaining observations that previously seemed incongruous.Accessible-surface modifications in the traditional manner add further variables to the studyof the ways in which analyte molecules interact with the mobile and stationary phaseson their "random walk" through the chromatographic bed.
Results of experiments using both bonded-silica phases and new bonded-hybrid particle phases,allied with recent ideas from related disciplines, will be shown to challenge traditionalconcepts of accessible surface, ligand density, silanol interaction, and hydrophobic "collapse".Particular attention will be paid to the role of the structure and physicochemical propertiesof both particle substrate and mobile phase elements as they, together,determine the constitution and function of the "stationary phase".
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
3Abstract
N
10 20 30 40 50 60 70 80 90 min
Unexpected
Note Peak Shape
Expected
4Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
1980 Curiosity Redux Discovery made by NOTwaiting for dry column
to equilibrate.Same result 20 yrs later:
Column: 3.9 x 150 mm; few pores > 100ÅSol-Gel Silica; fully C18 bondednot endcapped; air-dried > 1 yr
Mobile Phase: 0.5 mL/min70% MeOH [good wetting]
Injections: made every 1.5 min, startingas soon as 1st column volume emerges
Quinoline[weak base]
10 20 30 40 50 60 70 80 min
N
5Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
No Loss of EfficiencyColumn: Similar, except dried 18-20 hrs,argon stream, 80-90°; injections 3 mins apart
• Super-dry column• Quinoline peak
shape degradationis NOT due toloss of efficiency.
• Constant tR fornaphthaleneindicatesNO changein phase ratio.
• Analyte-accessiblepores wet quickly.
6
10 20 30 40 50 60 70 80 min
N
Column: High Purity Synthetic Silica GelC18, endcapped; more surface area in larger pores4.6 x 150 mmdried 18-20 hrs, argon stream, 80-90°
Injections: 3 mins apartMobile Phase: 70% MeOH, 0.7 mL/min
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Different Silica, Bonding
Subtle, steady asymmetryincrease for quinoline peak.
R2 = 0.9265
R2 = 0.9482
0.5
1.0
1.5
2.0
0 5 10 15 20 25 30 35
Quinoline
Naphthalene
Injection Number
Asymmetry
No loss of efficiency, constant tR for naphthalene.
How much of the surface in a porous particlecan be accessed by:• bonding reagents? • solvents? • analytes?
What is ligand density in bonded phase?Where are surface silanols? What’s their role?Can modifications to surface or substrate
structure be used to answer our questions?Traditional Cartoons are
misleading, implyingincomplete coverage of
accessible surface& direct analyte–silanol
access.
7Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Challenging Questions
O–SiO–SiOHO–SiO–SiOHO–SiOHO–SiO–SiO–SiOH
O–SiO–Si
O–SiO–SiO–
O–SiO–SiO–
O–SiO–
O–SiO–SiO–SiO–
O–SiO–Si
Mobile PhasepH > 3
Mobile PhasepH < 3
(CH3)2HN+
NH(CH3)2+
Surface
Core
Si
O
O O Si
O
O Si
O
O Si
O
O Si
O
O Si
O
OHOH
OSi
Si
O
O O
O O Si
O
O
Si
O
Si
O
Si
O
O Si
O
O O O O O O O
OH OH OH
SiO O Si O Si O Si O Si O Si OSiO O Si O Si
Bonded Silica Par ticle
8Ink bottles, craters, cylinders, plates are modelswhich lend themselves to simple math, BUT...
REALITY is Geodesic-domed labyrinths –formed withtetrahedral
building blocks.ALL surfaceshave terminal
–Si–OHgroups.
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Silica Pore Structure
OHSi
O OO
SiO
OHO
SiO
OHO
Si O
OO
SiO
SiO
OH
SiOH
O
SiO
O
O
Si
SiSi OH
O OSi O
OSi
SiSi
Si
Si
Si
SiHOSi
O
SiO
OSi
O
O
Si OHO O
Si Si
Si
SiOHO
OSi
SiO
HOO
SiO
HO
HO
Si
SiO
SiO
OO
Si
SiOH
O
OSi
OHOO
Si
Si
Si OH
OOSi
OO Si O
OSi
Si
SiSi
SiCH3
CH3
CH3
SiH3C
H3C
CH3
9Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Pore Access Level 1Most accessible
to largesilyl reagents
under favorablereaction
conditions;easiest-to-access
surface reactsfirst &
completely.
Outer surface & surfacein pore spaces > ~100 Åin diameter nearest tooutside of particle
OHSi
O OO
SiO
OHO
SiO
OHO
Si O
OO
SiO
SiO
OH
SiO
O
SiO
O
O
Si
SiSi OH
O OSi O
OSi
SiSi
Si
Si
Si
SiOSi CH3
H3C CH3
Si CH3
H3CCH3
SiO
SiO
OSi
O
O
Si OHO O
Si Si
Si
SiOHO
OSi
SiO
HOO
SiO
O
HO
Si
SiO
SiO
OO
Si
SiH3C
H3CH3C
SiOH
O
OSi
OHOO
Si
Si
Si O
OOSi
OO Si O
OSi
Si
SiSi
SiCH3
CH3
SiCH3
CH3
CH3
SiH3C
H3C
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Pore Access Level 2
Accessible tosmaller silylendcapping
reagents&
solventmolecules
Surface in pore spaces > ~50 Ådirectly connected to Level 1 pores.Diffusion distance fromouter surface is still minimal.
10
OHSi
O OO
SiO
OHO
SiO
OHO
Si O
OO
SiO
SiO
OH
SiO
O
SiO
O
O
Si
SiSi OH
O OSi O
OSi
SiSi
Si
Si
Si
SiOSi CH3
H3C CH3
Si CH3
H3CCH3
SiO
SiO
OSi
O
O
Si OHO O
Si Si
Si
SiOHO
OSi
SiO
HOO
SiO
O
HO
Si
SiO
SiO
OO
Si
SiH3C
H3CH3C
SiOH
O
OSi
OHOO
Si
Si
Si O
OOSi
OO Si O
OSi
Si
SiSi
SiCH3
CH3
SiCH3
CH3
CH3
SiH3C
H3C Porosity deeper
in the labyrinth,
accessible only
to small solvent
molecules;
hydrogen-bonding
molecules are most
favorably attracted,
esp., water.
Infusion of water
into these pores can take minutes.
11Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Least Accessible Level 3
12aPoster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Surface CoverageC = % Carbon [from CH analysis]
MWs = Mol. Wt. of silyl functionSC = mole % Carbon in silyl functionMs = Micromoles of Silyl FunctionSA = Surface Area Accessible to Chlorosilane [in sq. meters/gram]
Ms/m2 = C × 106
(SC – C) × MWs × SA
in µmoles/square meter
Ms/m2 = C × 106
(77.4 – C) × 310 × SA
For dimethyloctadecylsilyl:
Ms/m2 = C × 106
(50 – C) × 72 × SA
For trimethylsilyl:
* GE Berendsen, L de Galan, J Liq Chromatogr 1(4) (1978) 403-426; ** A Ulman, Adv Mater 2(12) (1990) 573-582
Accessible Surface Ligand Density May Reach Theoretical Maximum:
Bonding One Silanol Group/1 nm2 = 1.66 µmoles/m2 of coverage.
Density of 4.8 Si–OHs/1 nm2 [estimated from crystal models*]
means maximum coverage is 8.0 µmoles/m2
In Self-Assembled Monolayers of Octadecyltrichlorosilane on silica wafers,
each molecule occupies ~ 0.2 nm2 **.
Does this imply that ~ 5 ligand molecules could bond
to all accessible Si–OH sites in 1 nm2 on a porous silica gel substrate?
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
12b
Recent Studies in Semiconductor FieldChallenge Myth that High Ligand
Densities on Silica SurfacesCannot Be Achieved:
“Very strong molecule–substrate interactions...result in... formation of chemical bonds...at the interface, molecules try to occupy every available binding site on thesubstrate...in this process they push together molecules that have already adsorbed,thus eliminating free volume....in all SA monolayers the spontaneous adsorptionat the organic material–substrate interface,together with the strong van der Waals attraction amongst the alkyl chains,are the driving force for the formation of highly ordered,and closely packed systems.”*
* A Ulman, Adv Mater 2(12) (1990) 573
High Ligand Density
0
10
30
50
70
90
10 100 1000
Cumulative Percentof Surface Area
Treated Ppt Silicate
BJH Desorption Average Pore Diameter, Å
93% > 50 Å74% > 100 Å50% > 120 Å
0
10
20
30
40
50
60
70
80
90
10 100 1000
Cumulative Percentof Surface Area
Synthetic Silica GelFully RP Bonded Unbonded
BJH Desorption Average Pore Diameter, Å
84 % > 50Å50 % > 70Å 3 % > 100 Å
170 m2/gavg. 65 Å
87 % > 50Å50 % > 100Å
340 m2/gavg. 91 Å
13Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
0
10
30
50
70
90
10 50 100 1000
Cumulative Percentof Surface Area
Synthetic Silica Gel
BJH Desorption Average Pore Diameter, Å
87% > 50Å50% > 100Å
0
10
30
50
70
90
10 50 100 1000
Cumulative Percentof Surface Area
Si–CH3 Hybrid
BJH Desorption Average Pore Diameter, Å
99+% > 50 Å70% > 100 Å50% > 120 Å
% Surface Accessible?Distribution of surface area vs pore diameter varies with silica type
& method of preparation. Pore size & diffusion distance limitaccess. Compare % Surface Area in Pores > 100 Å [ ]*.
0
10
30
50
70
90
10 100 1000
Cumulative Percentof Surface Area Sol-Gel Silica
BJH Desorption Average Pore Diameter, Å
94% > 50 Å3% > 100 Å50% > 83 Å
Avg Pore Diameter isreduced by ~ length ofall-trans silyl function.
Bonding occursin larger pores.Micropores are
blocked.Surface area
cut in half, mostlyin pores >100Å
* All silicas used in these studies were made in our laboratories.
14a
0 40 min
Vo
Retention Times Stable:77 Injections, 20 hrs
Stopped Flow > 10 minRestart Flow: Retention Lost
100% aqueous mobile phase
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Wetting Pores‡
‡T Walter, et al, Poster, HPLC’97, [Waters Applications Library #980947, http://www.waters.com/pdfs/TWHPLC97.pdf]
Water on C18
γ = 72.8 dynes/cmθ = 110.6°*Pc ~ + 2200 psi
Methanol on C18
γ = 22 dynes/cmθ = 39.9°*Pc ~ –1500 psi
Pc = – cos θ4γd
d = capillary or pore diameterγ = suface tensionθ = contact anglePc = capillary pressure
θ > 90°cos θ is –
θ~110°d 67Å
θ~40°
θ < 90°cos θ is +
d 67Å
Observation: Explanation: Young-Laplace Theory
* B Janczuk, T Bialopiotrowicz, W Wojcik, Colloids Surfaces 36 (1989) 391-403
Organic-water
mixturesrequire Pc
between theseextremes.Column
Length is acritical
variable inwetting.
Column Void Volume1 1.2 1.4
0
20
60
100
1 1.2 1.4 mL
% Dewetting
2.0 µmol/m2 C18R2 = 0.825 N = 9
y = 263 + -188x
3.2 µmol/m2 C18R2 = 0.984 N = 4
y = 421 + -340x
28 17 0
t0
Decrease in Vo
correlates with% Dewetted
These data suggest that:as stationary phase dewets,mobile phase is extruded from pores.Hydrophobic Collapse is a MYTH!
Vo Decreases
14b
C18 C8 0
20
40
60
80
100% Dewetting after Stopping Flow
100% Aqueous95% Aqueous90% Aqueous
Mobile Phases:
RP[n=12]
RP[n=8]
embedded polar groupin Carbamate RP Phases
–O–Si–(CH2)3–O–C–NH–CnH2n+1
O
CH3
CH3
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Polar Group Effect
‡T Walter, et al, Poster, HPLC’97, [Waters Applications Library #980947, http://www.waters.com/pdfs/TWHPLC97.pdf]
Less dewetting with anembedded polar group,even with:• high ligand density• small pore size [65Å]
*
* U Neue, C Niederländer, J Petersen,U.S. Patent #5,374,755 (1994)
Proton Transfer AnalogyBR-StateProton Path
ProtonPickup
http://www.worthpublishers.com/lehninger3d/index.html
15aPoster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
PROTON PUMP:
Protons move
rapidly through
hydrophobic
regions of
bacteriorhodopsin
via a
proton path or
conduit
formed by
hydrogen-bonded
moieties in protein
structure & water
molecules.Proton Release
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
A Proton Pump
http://www.worthpublishers.com/lehninger3d/index.html
15b
16Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Protons in Water Wire
12
34
56H3O+
12
34
56
H5O2+
12
34
56
12
34
56
H3O+
12
34
56
H3O+
12
34
56
H5O2+
12
34
56
H3O+
Time: 0 fsecsTime: 30 fsecs
Time: 120 fsecsTime: 150 fsecs
Time: 200 fsecsTime: 230 fsecs
Time: 280 femtoseconds RR Sadeghi & H-P ChengJ Chem Phys 111(5) [1999] 2086-2094
Proton Transfer is one of the fastest processes in solution.Protons hop back & forth through “Water Wires”, by means of
rapid H-bond length fluctuations.
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
12
34
56HA
B
12
34
56A-
BH+
12
34
56A-
B
12
34
56A-
H3O+ B
Time: 0 fsecs
Time: 940 fsecsRR Sadeghi & H-P Cheng
J Chem Phys 111(5) [1999] 2086-2094
• Relative O–H
orientation important
• Momentum is crucial
for movement in a
given direction
• Sensitive to temperature
• Time scale is rapid!
– sub-picosecond
Base Creates Momentum 17
HydroLinked Proton Conduit™
* RR Sadeghi & H-P Cheng, J Chem Phys 111(5) [1999] 2086-2094** LF Scatena, MG Brown & GL Richmond, Science 292(4 May) [2001] 908-912
HLPC ConceptPoster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Two Physical Conditions Requiredfor Water Wire Formation*:
1. Weak Interaction betweenwater molecules & surroundings
2. Single strand of water moleculesresponsible for conduction of protons
18
Vibrational studies show that dipolar interactions betweenweakly hydrogen-bonded water molecules and hydrophobic surfaces
cause strong orientation effects in the interfacial region. This has“important implications” for a molecular-level understanding of “some
of the most important technological and biological processes.”**
OHSi
O OO
SiO
OHO
SiO
OHO
Si O
OO
SiO
SiO
OH
SiO
O
SiO
O
O
Si
SiSi OH
O OSi O
OSi
SiSi
Si
Si
Si
SiOSi CH3
H3C CH3
Si CH3
H3CCH3
SiO
SiO
OSi
O
O
Si OHO O
Si Si
Si
SiOHO
OSi
SiO
HOO
SiO
O
HO
Si
SiO
SiO
OO
Si
SiH3C
H3CH3C
SiOH
O
OSi
OHOO
Si
Si
Si O
OOSi
OO Si O
OSi
Si
SiSi
SiCH3
CH3
CH3
SiCH3
CH3
CH3
SiH3C
H3C
CH3
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
MeOH-Water Wire 19Not only does the
organic mobile phase componentfacilitate ‘wetting’ the pores.
Protic solvent moleculessuch as methanol can formlinks in the proton conduit.
OHSi
O OO
SiO
CH3O
SiO
OHO
Si O
OO
SiOH3C
SiO
O
SiCH3
O
SiO
O
O
Si
SiH3C
Si CH3
O OSi O
OSi
SiH3C
SiSi
SiCH3
Si
SiO
Si CH3H3C CH3
SiH3CO
SiO
OSi
O
O
Si OHO O
Si SiCH3
Si
SiOH3C
OSi
SiO
HOO
SiO
O
H3C
Si CH3
SiO
SiO
OO
Si
SiH3C
H3CH3C
SiOH
O
OSi
CH3OO
Si
SiH3C
Si O
OOSi
OO Si O
OSi
Si
SiH3C Si CH3Si CH3
O
NH
SiCH3
CH3
CH3
SiH3C
H3C
CH3
O O
NHO
SiCH3H3C CH3
20Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
RP Hybrid* PhaseThroughout
backbone,one third of
–Si–OH groupsare replaced
with –Si–CH3.Hydrophobicity
is increasedin micropores
as well asin mesopores
& macropores.
Polar embeddedcarbamate group**
* Patent Allowed** U Neue, C Niederländer, J Petersen, U.S. Patent #5,374,755 (1994)
21
~Si–OH ~Si–O-0
k20
40
60
3 pH5 7 9 11
Si–CH3
HybridSilica GelPolymer
Sol-GelSilica
Silica GelPolymer
Amitriptyline pKa ~ 9.4 [water] [8.5 [20% ACN] ]NH(CH3)2+
N(CH3)2
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
Compare SilicasRetention of strongly
basic analyte reaches amaximum at same pH
for two differentkinds of silica.Hybrid silica is
clearly different:hydrophobicity
within pores maintainsk at a higher level at
higher pH. Acidity ofsilanols is different -
smaller population ofSi–OH functions may
reduce opportunity forH-bonding between
adjacent silanols.
OHSi
O OO
SiO
CH3O
SiO
OHO
Si O
OO
SiOH3C
SiO
O
SiCH3
O
SiO
O
O
Si
SiH3C
Si CH3
O OSi O
OSi
SiH3C
SiSi
SiCH3
Si
SiO
Si CH3H3C CH3
SiH3CO
SiO
OSi
O
O
Si OHO O
Si SiCH3
Si
SiOH3C
OSi
SiO
HOO
SiO
O
H3C
Si CH3
SiO
SiO
OO
Si
SiH3C
H3CH3C
SiOH
O
OSi
CH3OO
Si
SiH3C
Si O
OOSi
OO Si O
OSi
Si
SiH3C Si CH3Si CH3
O
NH
SiCH3
CH3
CH3
SiH3C
H3C
CH3
O O
NHO
SiCH3H3C CH3
Protons hopwithinwater layer,instead ofmovingtowardbasicanalyte.Peakshapeimproves.
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
RP Ligand Disrupts Wire
Hybrid ParticleInhibits Wire
FormationHydrophobicitybuilt into hybridparticles’ pores
lowers attraction forwater molecules.
Water wiresless likely to form.
22
23Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
New Notions - Summary• Pore surface area accessibility is key to effective
surface modification & RP HPLC performance.• Ligand density may reach maximum in accessible
pores – total surface area calculation is misleading.• Pore dewetting, not hydrophobic collapse,
causes retention loss.• Silanols, even in micropores, may affect analytes
via water wires.• Water wires may be disrupted by embedded polar
groups, inhibited by hybrid particle pores.• Hybrid particle technology holds promise for
stationary phase performance improvements.
24aWaters Authors•Observations on the Wetting of HPLC Packings, T Walter, P Iraneta, M Capparella, Poster #P-202/A, HPLC’97, Birmingham
(1997) 19 pp [http://www.waters.com/pdfs/TWHPLC97.pdf or Search Waters Applications Library for 980947]•Dependence of cyano bonded phase hydrolytic stability on ligand structure and solution pH, JE O’Gara, BA Alden, CA
Gendreau, PC Iraneta, TH Walter, J Chromatogr A 893(2) (2000) 245-251•Systematic Study of Chromatographic Behavior vs Alkyl Chain Length for HPLC Bonded Phases Containing an Embedded
Carbamate Group, JE O’Gara, DP Walsh, BA Alden, P Casellini, TH Walter, Anal Chem 71(15) (1999) 2992-2997•Improving Our Understanding of Reversed-Phase Separations for the 21st Century, PD McDonald et al., Lecture #154,
ISC 2000, London, 3 Oct 2000Proton Transfer & Protein Pumps, Hydrogen Bonding in Water•The Dynamics of Proton Transfer in a Water Chain, RR Sadeghi, H-P Cheng, J Chem Phys 111(5) (1999) 2086-2094•Lehninger Principles of Biochemistry, 3rd ed, http://www.worthpublishers.com/lenhinger3d/iindex.html•Comment on the mechanism of proton-coupled electron transfer reactions, S-I Cho, S Shin, J Molecular Structure 499 (2000)
1-12•Time-Resolved Dynamics of Proton Transfer in Proteinous Systems, M Gutman, E Nachliel, Annu Rev Phys Chem 48 (1997)
329-356•Biophysical aspects of intra-protein proton transfer, S Brandsburg-Zabary, O Fried, Y Marantz, E Nachliel, M Gutman, Biochim
Biophys Acta 1458 (2000) 120-134•Ab initio analysis of proton transfer dynamics in (H2O)3H
+, PL Geissler, C Dellago, D Chandler, J Hutter, M Parrinello, ChemPhys Lett 321 (2000) 225-230
•Interactions of Hydration Water and Biological Membranes Studied by Neutron Scattering, J Fitter, RE Lechner, NA Dencher, JPhys Chem B 103 (1999) 8036-8050
•A direct-dynamics study of proton transfer through water bridges in guanine and 7-azaindole, Z Smedarchina, W Siebrand, AFernández-Ramos, L Gorb, J Leszczynski, J Chem Phys 112(2) (2000) 566-573
•Oxygen and Proton Pathways in Cytochrome c Oxidase, I Hofacker, K Schulten, PROTEINS: Structure, Function, & Genetics30 (1998) 100-107
•The dynamics of hydrogen bonds and proton transfer in zeolites - joint vistas from solid-state NMR and quantum chemistry, HKoller, G Engelhardt, RA van Santen, Topics in Catalysis 9 (1999) 163-180
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
References 1
24b•How water takes shape at hydrophobic surfaces, GL Richmond, C&EN, Sept 11 (2000) 29•Autoionization in Liquid Water, PL Geissler, C Dellago, D Chandler, J Hutter, M Parrinello, Science 291 (16 Mar 2001) 2121-
2124•An Introduction to Hydrogen Bonding, GA Jeffrey, Oxford University Press, New York (1997) pp 119-123•The Structure and Properties of Water, D Eisenberg, W Kauzmann, Oxford University Press, New York (1969) pp 225-227
Monolayers, Surfaces, Pores•Self-Assembled Monolayers of Alkyltrichlorosilanes: Building Blocks for Future Organic Materials, A Ulman, Adv Mater 2(12)
(1990) 573-582•Self-Assembled Mono- and Multilayers of Terminally Functionalized Organosilyl Compounds on Silicon Substrates, S Heid, F
Effenberger, Langmuir 12 (1996) 2118-2120•Formation of Uniform Aminosilane Thin Layers: An Imine Formation to Measure Relative Surface Density of the Amine Group,
JH Moon, JW Shin, SY Kim, JW Park, Langmuir 12 (1996) 4621-4624•Pore-Resolved NMR Porosimetry, RS Drago, DC Ferris, DS Burns, J Am Chem Soc 117 (1995) 6914-6920•The Surface Tension Components of Aqueous Alcohol Solutions, B Janczuk, T Bialopiotrowicz, W Wojcik, Colloids Surfaces
36 (1989) 391-403
Reversed-Phase HPLC•Solvophobic Interactions in Liquid Chromatography with Nonpolar Stationary Phases, C Horváth, W Melander, I Molnár, J
Chromatogr 125 (1976) 129-156•The Molecular Mechanism of Retention in Reversed-Phase Liquid Chromatography, JG Dorsey, KA Dill, Chem Rev 89 (1989)
331-346•Retention in reversed-phase chromatography: partition or adsorption?, A Vailaya, C Horváth, J Chromatogr A 829 (1998) 1-27•Revisionist look at solvophobic driving forces in reversed-phase liquid chromatography, PW Carr, J Li, AJ Dallas, DI Eikens, LC
Tan, J Chromatogr A 656 (1993) 113-133
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
References 2
24cSilica & Bonded Phases•Porous Silica, KK Unger, J Chrom Library, 16 Elsevier (1979) 359 pp•Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized
high-resolution chromatographic and electrokinetic separations, KK Unger, D Kumar, M Grün, G Büchel, S Lüdtke Th Adam,K Schumacher, S Renker, J Chromatogr A 892 (2000) 47-55
•A Geometrical Model for Chemically Bonded TMS and PDS Phases, GE Berendsen, L de Galan, J Liq Chromatogr 1(4) (1978)403-426
•Preparation and Chromatographic Properties of Some Chemically Bonded Phases for Reversed-Phase Liquid Chromatography,GE Berendsen, L de Galan, J Liq Chromatogr 1(5) (1978) 561-586
•Preparation of Various Bonded Phases for HPLC Using Monochlorosilanes, GE Berendsen, KA Pikaart, L de Galan, J LiqChromatogr 3(10) (1980) 1437-1464
•Determination of Bonded Phase Thickness in Liquid Chromatography by Small Angle Neutron Scattering, LC Sander, CJGlinka, SA Wise, Anal Chem 62(10) (1990) 1099-1101
•Geometry of Chemically Modified Silica, I Rustamov, T Farcas, F Ahmed, F Chan, R LoBrutto, HM McNair, YV Kazakevich, JChromatogr A [HPLC 2000 Proceedings Volume] in press
•Interpretation of the Excess Adsorption Isotherms of Organic Eluent Comoponents on the Surface of Reversed-PhaseAdsorbents. Effect on the Analyte Retention, YV Kazakevich, R LoBrutto, F Chan, T Patel, J Chromatogr A [HPLC 2000Proceedings Volume] in press
•Chromatographic silanol activity test procedures: the quest for a universal test, SD Rogers, JG Dorsey, J Chromatogr A 892(2000) 57-65
Other key references cited in footnotes on individual pages.
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
References 3
http://www.waters.com; click on Applications Library under HPLC Columns or Applications Headings
Poster by Patrick D. McDonald, Ph.D., HPLC 2001, Maastricht, 18-19 June 2001. © 2001 Waters Corporation All photos © 2001 by PDMcD. Waters, µBondapak, Symmetry, XTerra, HLPC, HydroLinked Proton Conduit are trademarks of Waters Corporation.
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