Carr Group Research

8/13/2019 Carr Group Research

1/17

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


2/17

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.


3/17

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


4/17

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


5/17

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


6/17

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


7/17

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


8/17

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


9/17

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

+


10/17

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


11/17

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)


12/17

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


13/17

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 . )


14/17

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


15/17

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


16/17

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


17/17

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

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