carr group research
TRANSCRIPT
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Ultra-Stable Stationary Phases for HPLC:
Assembly, Advantages, and Applications
Lianja Ma, Dwight Stoll, Hao Luo, Adam Schellinger, XiaoliWang, Yu Zhang, Chang Yub Paek and Peter W. Carr*
Peter W. Carr GroupChemistry Department
University of Minnesota
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Group Goals
Demonstrate advantagesChemical and thermal stability
Synthesize highly stable stationary phases for RPLCSilica-based hypercrosslinked reversed and ion exchange phases
ApplyUltra- Fast High Temperature Liquid Chromatography
(UFHTLC )
Two- Dimensional HP LC (2DLC )
Fast gradient elution chromatography of forensic samples.
Optimization of gradient elution peak capacity for proteomicsstudies.
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What are the Advantages of Highly StableStationary Phases?
Allows Cleaningwith Conc. Acid
Ion Suppressionof COOH
pH < 1
SanitizationDepyrogenation
Ion Suppressionof NH2
pH >13
pHStability
Less Wareand Tare
Faster analyses
Higher Flow
Rate
Lower Pressure
Drop
LessOrganicSolvent
MoreRobust
Analysis
Easier MethodDevelopment
ThermallyOptimizedSelectivity
ThermalStability
Extraordinary ChemicalStability
Save $$
GreenChemistry
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Hyper-Crosslinked (HC) Platform Prepared byOrthogonal Friedel-Crafts Chemistry
Cl
Cl
ClCl
ClCl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H2CCl
SiCH 3 CH 3Cl
a b
Crosslink with
Styrene Heptamer
(SH)
SH CrosslinkedDM-CMPES
SecondarilyCrosslinkedDM-CMPES
Secondary
Crosslink with
(Chloromethyl)methylether(CH 3OCH 2Cl)
ClCl
Cl
Cl
Cl
Cl
Cl
Cl
DiMethyl ChloroMethylPhenylEthylSilane(DM-CMPES)
b: Styrene Heptamer a: DiMethyl-Chloro
MethylPhenylEthylSilane(DM-CMPES)
Amplified View of APorous Silica Particle
Surface
Friedel-Crafts reactions catalyst: SnCl 4
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Synthesis of HC-C 8 and -SO 3-HC-C 8
SecondarilyCrosslinkedDM-CMPES
Cl
Cl
HC-C 8
1-octylbenzene
ClCl
Cl
Cl
Cl
Cl
Cl
Cl
ClSO 3H
Alkyl chain
Cl
Cl
S O 3 -
S O 3 -
S O 3 -
-SO 3-HC-C 8
- S O
3 -
- S O 3
-
- S O 3 -
CationExchangeSite
A novel platform for a family of new phaseswith different
separation modes
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Column volume
0 500 1000 1500 2000 250050
60
70
80
90
100
0.1/49.95/49.95 TFA/ACN/water pH = 2.0Temp = 150 CFlow rate: 0.5 mL/minSolute: Hexadecanophenone
Ultra High Acid Stability of HC-C 8
Very good acid stability is achieved by the formation of hyper-crosslinked polymer networks
HC-C 8 before HF digestion
HC-C 8 After HF digestionall silica was removed
Acid stability at pH 2 and 150C
Column: 3.3 0.21cm49.95/49.95/0.1 ACN/Water/TFA , 150 C, 0.5 mL/min
Solute: hexadecanophenone
SB C 18
HC-C 8
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Basic Drugs Performance Comparison ofSB C 18 and HC-C 8 in Formic Acid
Time (min)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
A b s o r b a n c e
( m A U )
0
2
4
6
8
10
Alprenololk' = 1.31
N = 900 Nortriptylinek' = 3.74
N = 3550
Amitriptylinek' = 4.28 N = 2750
SB C 18
Time (min)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
A b s o r
b a n c e
( m A U )
0
2
4
6
8
10
Alprenololk' = 0.93
N = 2450
Nortriptylinek' = 3.36
N = 4150
Amitriptylinek' = 4.04
N = 3800
HC-C 8
HC-C 8: 34/66 ACN/water, SB C 18 38/62 ACN/water. For both columns: 0.1% formic acid , 40C, 1mL/min
5 0.46 cm column
HC-C 8 provides excellent efficiency for basic drugseven in weak ion pairing reagent formic acid
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Temperature
The Third Dimension in HPLCTemperature
Mobile Phase Stationary Phase
Applications:Affects speed - UFHTLC
Affects selectivity T3C
Limitations:Stationary phase stability
Analyte stabilityThermal mismatch broadening
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Ultra-Fast High Temperature LiquidChromatography (UFHTLC) - Effect of
Temperature on Analysis Time
High-Performance Liquid Chromatography atElevated Temperatures: Examination of
Condition for the Rapid Separation of LargeMolecules, R. D. Antia and Cs. Horvath, J.Chromatogr ., 435, 1-15 (1988).
Applications of UFHTLCDramatically increases throughput for
routine analyses, decreasing totalanalysis costIncrease screening rate in combinatorialchemistry (speed up LC side of LC-MS)
Make 2D-HPLC practical and thusgreatly enhance resolving power ofHPLC
T ( oC)
40 60 80 100 120 140 160 180 200
t a n a
l y s i s (
T ) / t a
n a l y s i s
( 2 5
o C )
0.0
0.2
0.4
0.6
0.8
1.0
3/13/2max
3/2)'1(
3/2 T P Lk
N t
Ah
+
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Ultra-Fast Separation of Alkylphenones
u = 3.3 cm/s ( = 150)
k max = 4.6
N = 1400n c = 8
P = 300 bar0
20
406080
100120
140160
0 5 10 15Time (sec.)
m A U
2
65
43
1
Column: 50 mm x 2.1 mm i.d. PBD-C-ZrO 2
Temperature: 150 oC
Flow rate: 4.75 ml/min.
Injection volume: 1 l
Detection at 254 nm with 6 l flow cell and 50 ms detector response time
Solutes: Acetone, propiophenone, butyrophenone, valerophenone, hexanophenone,and heptanophenone
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Worlds Fastest Gradient Elution RPLC
-25
125
275
425
0 10 20 30 40 50 60 70
Tim e (sec.)
m
A U
0.0
0.2
0.4
0.6
0.8
1.0
Column: SB300-C 18 50 mm x 2.1 mm i.d.Flow rate: 3.0 ml/min .Temperature: 100 oC
Gradient ConditionsA: 0.1% Trifluroacetic acid in water; B: 0.1% Trifluroacetic acid in ACNGradient from 0-100% B in 21 seconds
Gradient from 0-100% B in 21 secondsSolutes:Uracil,
Nitroalkanehomologs (2-5)
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Fast , Comprehensive Two-Dimensional HPLC An Approach to Dramatically Increasing the
Resolving Power of HPLCComprehensive two-dimensional
HPLC can dramatically increase total peak capacity
( )1'ln4
1 ++= ns
c k R N
n
One-dimensional separations in HPLCare limited by low peak capacity
21 cccTotal nnn =
1 2
3 4
5
6 7 8
9
1 0
5000
17000
290000
10
20
30
40
50
60
P e a
k C a p
c i t y
( n c
)
Retention Factor (N)
( ) ( )[ ])2
2max211max 1'1'
++=
k L N k t crtotal
A major limitation, however, is theslow speed, which is related to the
second dimension linear velocity, u 2
Giddings, J. C. Multidimensional Chromatography: Techniques and Applications ; Marcel Dekker: New York, 1990
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2DLC Separation of Corn Seedling Extract> 200 Peaks in 30min
F i r s t D i m e n s i o n R e t e n t i o n T i m e ( m i n . )
mAU
S e c o n d
D i m e
n s i o n
R e t e
n t i o n T
i m e (
s e c . )
F i r s t D i m e n s i o n R e t e n t i o n T i m e ( m i n . )
mAU
S e c o n d
D i m e
n s i o n
R e t e
n t i o n T
i m e (
s e c . )
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Two Dimensional Separations are Rich with Information
0
500
1000
1500
2000
2500
3000
0 5 10 15 20 25 30
Time (min.)
m A U
0
10
20
30
40
50
60
0 5 10 15 20
Second Dimension Retention Time (sec.)
m A U
At least nine peaks are observed in thesecond dimension from single firstdimension peak (9.80-10.15 min.)
I d P k C it B gi t Mitig t th D i
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Increased Peak Capacity Begins to Mitigate the DynamicRange Problem that Plagues Bioanalytical Separations
0
500
1000
1500
2000
2500
3000
0 5 10 15 20 25 30
Time (min.)
m A U
0
5
10
15
20
25
30
35
40
0 5 10 15 20
Second Dimension Retention Time (sec.)
m A U
Several low abundance species are
detected in the 2DLC separation thatwould otherwise be obscured by high
abundance peaks in a one-dimensionalseparation
d k d d
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Optimized Peak Capacity Production in GradientElution Separation of Peptides Separation
Time (min)
0 20 40 60 80 100 120
A b s
o r b a n c e
( m A U )
0
10
20
30
40
Chromatographic Conditions:
Five Poroshell 300SB-C18 columns connected in series, 2.1mm i.d., 5 m, L = 60 cm
Solvent A: 0.1% TFA in H 2O, Solvent B: 0.1% TFA in 80:20 ACN:H 2O
Gradient: 0 40 100 0 %B at 0 120 160 200 min, Pressure = 315 bar
0.50 mL/min, 70 oC, 5 L injection, 13 L flow cell, 214 nm, HP 1100
Retention window = 180.8 min, Average peak width = 0.231 min
Peak capacity = ~ 500
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Conclusions
Highly crosslinked silica phases are VASTLY more stable
in acid media than sterically protected ODS phase UFHTLC dramatically increases throughput for routine
analyses, decreasing total analysis time and cost
Fast gradient elution allows rapid identification
LC UFHTLC is a very useful approach to enhance the
peak capacity of HPLC